( E ) Results of MS analyses of affinity co - purification experiments in wild - type B . subtilis control ( BTM2 , Table EV2 ) B . subtilis deficient in endogenous YqgP expressing wild - type YqgP - sfGFP bait ( BTM84 , Table EV2 ) or proteolytically inactive YqgP . S288A - sfGFP bait ( BBM1 , Table EV2 ) . were considered as potential interactors of YqgP if they were identified only in both positive co - purifications with a minimum of 3 weighted spectral or enriched at least 5 times in positive bait samples compared to control ones on weighted spectral . highlighted in red was only overlapping hit between two proteomics approaches .

(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.


( A ) Steady - state cleavage profile of endogenous MgtE processed by endogenous ectopically overexpressed YqgP from inducible Phyperspank promoter in living B . subtilis ( BTM2 BTM501 , respectively , Table EV2 ) , in minimal medium at low magnesium ( 10 µM ) . lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) was used as a control . were detected by immunoblotting with chemiluminescence detection . Data information : In panels , endogenous full - MgtE is indicated by a black arrow , N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)


( B ) cleavage is efficiently inhibited by 1 μM STS736 , a specific peptidyl ketoamide rhomboid inhibitor Ticha et al . , 2017b ( ) . Data information : In panels , endogenous full - MgtE is indicated by a black arrow , N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) ectopic YqgP by anti - YqgP antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( C ) To map cleavage site region within endogenous MgtE ( second lane from left , from BTM501 ) , MgtE - derived reference fragments encoding first 300 , 315 , 330 as well as full - MgtE ( black arrow ) , were in vitro transcribed translated . Mobility of N - terminal cleavage product ( red arrow ) of MgtE on SDS PAGE was compared to mobilities of translated reference fragments . Data information : In panels , endogenous full - MgtE is indicated by a black arrow , N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) ectopic YqgP by anti - YqgP antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( A ) Detection of cleavage of endogenous MgtE by YqgP in living B . subtilis ( BS72 , Table EV2 ) depending on concentrations of magnesium manganese ions . were cultivated in glucose M9 minimal medium with limiting ( 0 . 01 mM ) or high ( 1 mM ) of MgSO4 , in presence or absence of 100 μM MnCl2 , analysed by western blotting with near - infrared detection ( upper panel ) . Black arrow denotes full - MgtE , red arrow denotes its N - terminal cleavage product formed by YqgP . corresponding signals were quantified by densitometry , are displayed as relative specific activity , which is substrate conversion normalized to enzyme expression level ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).


( B ) Growth curves of wild - type ( BTM843 , Table EV2 ) , yqgP deficient ( BTM844 , Table EV2 ) rescue ( BTM845 , Table EV2 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese stress elicited by adding 75 µM MnSO4 in mid - exponential phase ( stress - phase denoted by blueish background ) . strains further contain a deletion in putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in yqgP deficient than in wild type , that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between strains in absence of manganese stress . Data are shown as datapoints from three independent experiments overlaid with dashed connecting average values from each time - point , which illustrates reproducibility of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.


( C ) Top panel : growth curves of wild - type ( BTM843 ) , yqgP deficient ( BTM844 ) rescue ( BTM845 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese shock elicited by adding 75 µM MnSO4 in mid - exponential phase . Bottom panel : manganese toxicity is prevented by further adding 5 mM magnesium ( MgSO4 ) in otherwise identical conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.


( D ) Inhibition of YqgP by a rhomboid - specific peptidyl ketoamide inhibitor ( STS736 , i . e . compound 9 from Ticha et al . , 2017b ( ) ) abolishes YqgP induced fitness of B . subtilis under manganese stress , in a dose - dependent manner , both with endogenous YqgP ( top panel ) overexpressed YqgP ( bottom panel ) . Media growth conditions were identical to those used in panel ( B ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).


( E ) Overexpression of heterologous MgtE inhibits growth of yqgP deficient ( BTM610 , Table EV2 ) in rich LB medium supplemented with 75 μM MnSO4 . fitness is improved by overexpression of YqgP ( BTM611 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BTM612 , Table EV2 ) . For clarity , for panels C - E , representative experiments of 2 - 3 independent biological replicates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.


( B ) Detection ( left panel ) ( right panel ) of steady - state conversion of endogenous MgtE model chimeric substrate derived from Providencia stuartii TatA Stevenson et al . , 2007Ticha et al . , 2017aTicha et al . , 2017b ( , , ) ( schemes in middle ) by ectopically expressed YqgP its N - C - terminally truncated variants ( strains BS184 - 187 , Table EV2 ) in living B . subtilis grown in rich LB medium . Black arrows indicate full - substrates red arrows indicate cleavage products .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.


( C ) Similar of same strains but grown in minimal M9 medium ( with 10 µM MgSO4 ) supplemented with increasing concentrations of MnCl 2 . Black arrows denote full - substrate ( MgtE ) , red arrows denote cleavage products generated by YqgP . ( D ) Western blots of four independent experiments shown in panel ( C ) were quantified by near - infrared detection , quantified by densitometry results are displayed as fold activation of MgtE cleavage at 100 µM MnCl 2 relative to 1 µM MnCl 2 activity . Average values fours datapoints are plotted for each indicated YqgP variant .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl2 relative to 1 µM MnCl2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.


( A ) Detection ( left panel ) ( right panel ) of steady - state conversion of endogenous MgtE by YqgP ( BS72 , Table EV2 ) grown in minimal M9 medium containing 10 μM MgSO4 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 CaCl2 ( reference conditions ) with additions of 10 µM or 100 μM of a given divalent cation salt solution ( MnCl2 , ZnCl2 , CoCl2 , or NiCl2 ) , or 50 µM 500 μM for CaCl2 . Western blots were quantified by near - infrared detection ( left ) , quantified by densitometry displayed as relative specific activity ( graph on right ) , which is substrate conversion normalized to enzyme expression level . Black arrow denotes full - substrate ( MgtE ) , red arrow denotes N - terminal cleavage product ( s ) generated by YqgP . bands originate from same western blot identical treatment series .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.


( B ) Isothermal titration calorimetry of purified recombinant YqgPNTD selected divalent cations .

(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.


( C ) Cation toxicity assays their relationship to YqgP activity . Wild type B . subtilis ( BTM2 , Table EV2 ) , its variant lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) , rescue ectopically expressing YqgP ( BTM501 , Table EV2 ) were cultivated in minimal M9 medium containing 10 μM MgSO4 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 CaCl2 ( i . e . reference conditions ) , in mid - exponential phase were stressed by addition of either 70 µM MnCl2 , 500 µM ZnCl2 , 25 µM CoSO4 or 400 µM NiCl2 ( pale blue ) . YqgP activity specifically improved fitness during manganese zinc stress , while it had no effect on growth of cultivated in presence of high cobalt nickel concentrations . Representative experiments of 2 - 3 independent replicates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.


( E Detection of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 . Black arrow marks full - MgtE , red arrow marks its N - terminal cleavage product by YqgP .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.


F ) of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.


( A ) proteolytically dead mutant YqgP . S288A behanes as a pseudoprotease acts as a substrate adaptor of FtsH . Pattern of C - terminally truncated forms of endogenous MgtE formed in vivo in presence of ectopically expressed active YqgP ( BS72 , Table EV2 ) or its inactive S288A mutant ( BS73 , Table EV2 ) in rich LB medium in presence or absence of endogenous FtsH . Black arrow denotes full - MgtE substrate , red arrows denote its cleavage products generated by YqgP . While presence of faster migrating YqgP - dependent products ( full red arrows ) do not depend on presence of FtsH , slower migrating product formed in presence of YqgP . S288A ( open red arrow ) does , indicating that FtsH protease is responsible for alternative cleavage of MgtE . same arrow symbolics are valid throughout this figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.


( B ) Mapping of boundary of FtsH generated proteolytic product of MgtE . Immunoblot comparing SDS PAGE mobility of N - terminal fragment of endogenous MgtE generated by FtsH in presence of pseudoprotease version of YqgP ( catalytically dead YqgP . S288A , strains BTM78 , BTM502 , Table EV2 ) to mobilities of in vitro translated reference fragments corresponding to MgtE 1 - 340 , - 355 - 370 shows that YqgP . S288A acts as an FtsH adaptor , that FtsH processing of MgtE in presence of YqgP . S288A stops at around acid 355 , which is near or at end of TMH 3 of MgtE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE.


( D ) In vivo cleavage of endogenous MgtE in presence of ectopically expressed full - YqgP . S288A ( BS73 ) , YqgPΔNTD . S288A ( BS185 , Table EV2 ) or YqgPΔCTD . S288A ( BS186 , Table EV2 ) , in rich LB medium shows that YqgPNTD is necessary for FtsH - dependent processing of MgtE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE.


( E ) of in vivo kinetics of formation fate of MgtE cleavage products . B . subtilis strains expressing YqgP or its S288A mutant ( strains BTM78 ; 501 502 , Table EV2 ) in presence or absence of endogenous FtsH protease ( strains BTM795 , BTM796 BTM797 , Table EV2 ) were cultured in LB medium at early exponential phase , after having been expressing YqgP variants for 30 min , were either left grown untreated ( as a control with ongoing translation ) or treated with 20 μg / mL tetracycline to stop proteosynthesis ( translation shut - off ) . At given time intervals afterwards , cultures were analysed by α - MgtE western blotting with near infrared detection . Equal number ( judged by OD600 ) was loaded into each lane . Black arrow marks full - MgtE , red full arrows mark YqgP - dependent red open arrows mark YqgP . S288A - dependent cleavage product of MgtE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.


B . Representative flow cytometry dot plots of CD11b + CD11b + / Cripto + at day 2 , 3 5 after injury . PE ( Phycoerythrin / free channel ) . Data are mean ± ( n = 5 biological replicates ; P ≤ 0 . 00008 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at day 2, 3 and 5 after injury. PE (Phycoerythrin/free channel). Data are mean±SEM (n=5 biological replicates; P≤0.00008, Student's t-test).


C . Representative flow cytometry dot plots of CD11b + / F4 / 80 + / Ly6CHigh / Low CD11b + / Cripto + / F4 / 80 + / Ly6CHigh / Low at day 2 , 3 5 after injury . D . of F4 / 80 + / Ly6CHigh / Low in CD11b + CD11b + / Cripto + population from injured at indicated time points . Data represent mean ± ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at day 2, 3 and 5 after injury. D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


A . Representative pictures of immunostaining for F4 / 80 ( green ) in Control CriptoMy - LOF TA sections at day 2 ( top panel ) 5 ( bottom panel ) after injury . B . of F4 / 80 / damaged ( μm2 ) at day 2 ( top graph ) 5 ( bottom graph ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).


C . Representative pictures of immunostaining for CD206 in Control CriptoMy - LOF TA sections at day 2 ( top panel ) 5 ( bottom panel ) after injury , respectively . D - E . of CD206 + MPs per ( mm2 ) at day 2 ( D ) 5 ( E ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively. D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).


A . Representative flow cytometry dot plots showing of GFP + population in GFP - Control GFP - CriptoMy - LOF at day 2 ( top panel ) 5 ( bottom panel ) after injury . of GFP + expressing F4 / 80 in GFP - Control GFP - CriptoMy - LOF is shown at day 2 after injury ( middle panel ) . Data are mean ± ( n = 4 biological replicates ; P = ns ; Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative flow cytometry dot plots showing the percentage of GFP+ cell population in GFP-Control and GFP-CriptoMy-LOF muscles at day 2 (top panel) and 5 (bottom panel) after injury. Percentage of GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles is shown at day 2 after injury (middle panel). Data are mean±SEM (n=4 biological replicates; P=ns; Student's t-test).


B . qRT - PCR of pro - ( Nos2 , Mcp1 Tnfα ) anti - ( Arg1 , Fizz1 , Il10 , Il4rα Tgfβ ) inflammatory markers in GFP - CriptoMy - LOF GFP - Control MPs at day 2 5 after injury . Data represent mean ± of relative mRNA level normalized with Gapdh ( n ≥ 4 biological replicates ; * * * P < 0 . 001 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. qRT-PCR analysis of pro- (Nos2, Mcp1 and Tnfα) and anti- (Arg1, Fizz1, Il10, Il4rα and Tgfβ) inflammatory markers in GFP-CriptoMy-LOF and GFP-Control MPs at day 2 and 5 after injury. Data represent mean±SEM of relative mRNA level normalized with Gapdh (n≥4 biological replicates; ***P<0.001, Student's t-test).


C . Representative pictures of immunostaining for GFP ( green ) , CD206 ( red ) , pSMAD3 ( ) in GFP - Control GFP - CriptoMy - LOF TA sections at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x . D . of GFP + / CD206 ± / pSMAD3 ± distribution in TA sections from GFP - Control GFP - CriptoMy - LOF at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Data are mean ± ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative pictures of immunostaining for GFP (green), CD206 (red), pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; **P<0.01, Student's t-test).


E . Representative flow cytometry dot plots of F4 / 80 + / Ly6CLow population gated on GFP + from GFP - Control GFP - CriptoMy - LOF at day 5 after injury . Data are mean ± ( n = 3 biological replicates ; P = ns ; Student ' s t - ) .

E. Representative flow cytometry dot plots of F4/80+/Ly6CLow cell population gated on GFP+ cells from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are mean±SEM (n=3 biological replicates; P=ns; Student's t-test).


( F ) Smear plot of - seq data from GFP + / F4 / 80 + / Ly6CLow FACS sorted from GFP - Control GFP - CriptoMy - LOF at day 5 after injury . Data show expression level as Log2 fold - change ( FC ) [ Log2 ( GFP - Control / GFP - CriptoMy - LOF ) ] , against Log2 ( expression ) [ Log2 ( average of gene expression across samples ] , for each gene . blue lines correspond to LogFC of 1 - 1 . ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as Log2 fold-change (FC) [Log2(GFP-Control/GFP-CriptoMy-LOF)], against Log2(expression) [Log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and -1. (n=3 biological replicates).


B . Representative pictures of double immunostaining with Laminin ( green ) embryonic myosin heavy chain ( eMHC ; red ) at day 5 ( left panel ) 30 ( right panel ) after cardiotoxin ( CTX ) injection . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. Data information: Nuclei were counterstained with DAPI (blue).


C . Minimal Feret ' s diameter distribution of Control CriptoMy - LOF myofibers at day 5 ( top panel ) 30 ( bottom panel ) after CTX injection . Data are mean ± ( n = 5 biological replicates ; P = ns , Student ' s t - ) .

C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean±SEM (n=5 biological replicates; P=ns, Student's t-test).


E . Representative pictures of double immunostaining with Laminin ( green ) eMHC ( red ) at day 5 ( left panel ) 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue).


F . Minimal Feret ' s diameter distribution of Control

F. Minimal Feret's diameter distribution of Control and Cripto


B . Representative pictures of immunostaining of mdx - Control mdx - CriptoMy - LOF diaphragm sections with GFP ( green ) CD206 ( ; left panel ) , pSMAD3 ( red ; middle panel ) GFP pSMAD3 ( right panel ) . C - E . of GFP ( C ) , GFP / CD206 ( D ) GFP / pSMAD3 ( E ) positive per ( mm2 ) in mdx - Control mdx - CriptoMy - LOF diaphragms . Data are expressed as box plots displaying minimum , first quartile , median , third quartile maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F - G . Representative pictures of Picrosirius red of mdx - Control mdx - CriptoMy - LOF diaphragm sections ( F ) of picrosirius red ( G ) . Data are expressed as of stained are box plots displaying minimum , first quartile , median , third quartile maximum ( n = 5 biological replicates ; * P < 0 . 05 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).


H . Hydroxyproline ( HOP ) in mdx - Control mdx - CriptoMy - LOF diaphragm muscle sections . Data are expressed as HOP ( μg ) per muscle section volume ( mm3 ) as box plots displaying minimum , first quartile , median , third quartile maximum ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).


Representative of Laminin ( red ) immunostaining of mdx - Control mdx - CriptoMy - LOF diaphragm sections of centrally nucleated myofibers . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


Representative mdx - Control mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution of centrally nucleated myofibers . Data are expressed as mean ± ( n = 5 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 ; Student ' s t - )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test)


Representative mdx - Control mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution Variance coefficient of centrally nucleated myofibers . box plots displaying minimum , first quartile , median , third quartile

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution Variance coefficient of centrally nucleated myofibers. box plots displaying minimum, first quartile, median, third quartile and maximum


L - M . Representative of eMHC ( red ) immunostaining of mdx - Control mdx - CriptoMy - LOF diaphragm sections ( L ) of eMHC + myofibers per ( mm2 , M ) . Data are expressed as box plots displaying minimum , first quartile , median , third quartile maximum ( n = 5 biological replicates ; * P < 0 . 05 ; Student ' s t - ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


A . Representative pictures of CD31 ( red ) immunostaining of Control CriptoMy - LOF TA sections at day 5 ( left panel ) 30 ( right panel ) after single injury ( CTX - I ) . B - D . of CD31 + capillary number per ( mm2 ; B ) , average of capillary cross - sectional ( µm2 ; C ) of small ( < 20 µm2 ) large ( > 100 µm2 ) capillaries ( D ) in Control CriptoMy - LOF injured at indicated time points after single injury ( CTX - I ) . Data are mean ± ( n = 5 biological replicates ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ' s t - ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


E . Representative pictures of CD31 ( red ) immunostaining of Control CriptoMy - LOF TA sections at day 5 ( left panel ) 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F . Representative pictures of mdx - Control mdx - CriptoMy - LOF diaphragm sections immunostained with VEcad ( red ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of boxes is 3 . 5 x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


G - H . of CD31 + capillary number per ( mm2 ; G ) average of capillary cross - sectional ( µm2 ; H ) in Control CriptoMy - LOF injured at indicated time points after re - injury ( CTX - II ) Data are mean ± ( n = 6 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ' s t - ) .

G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).


I - J . of capillary number per ( mm2 ; I ) average of capillary cross - sectional ( µm2 ; J ) in mdx - Control mdx - CriptoMy - LOF diaphragm . Data are mean ± ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


A . Representative pictures of double immunostaining with VEcad ( red ; left middle panel ) KLF4 ( green , left panel ) or TCF4 ( green , middle panel ) , with CD31 ( red ; right panels , confocal pictures ) TCF4 ( , right panel ) on Control CriptoMy - LOF TA sections at day 5 after injury . B - D . of VEcad / KLF4 ( B ) , VEcad / TCF4 ( C ) CD31 / TCF4 ( D ) double positive per ( mm2 ) in Control CriptoMy - LOF TA sections at day 5 after injury . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).


E . Representative Confocal pictures of CD31 + ( red ) PDGFRα + ( green ) immunostaining on Control CriptoMy - LOF TA sections at day 5 after injury . F - G . of CD31 + / PDGFRα + ( F ) CD31 - / PDGFRα + ( G ) per ( mm2 ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).


H - I . Representative pictures of triple immunostaining with VEcad ( red ) , KLF4 ( green ) pSMAD3 ( ) on Control CriptoMy - LOF TA sections at day 5 after injury ( H ) of VEcad / KLF4 / pSMAD3 triple positive per ( mm2 ; I ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) KLF4 ( green ) on Control CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - II Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) KLF4 ( green ) on diaphragm sections of mdx - Control mdx - CriptoMy - LOF Data information : Nuclei were counterstained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).


of VEcad / KLF4 double positive per ( mm2 ) in Control CriptoMy - LOF TA sections at day 5 after re - injury ( CTX -

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II


of VEcad / KLF4 double positive per ( mm2 ) in diaphragm sections of mdx - Control mdx - CriptoMy -

O O O O O O O O O O O O O O O O O O O O O

Quantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF


A ROS were measured by flow cytometry after with specific oxidative stress detection dyes ( CellROX green , Molecular Probes , USA ) in HC ( n = 4 ) DM ( n = 4 ) platelets . ( * * p = 0 . 032 vs . HC ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).


B Western blot of ROS downstream signaling molecules p53 phosphorylated p53 in HC ( # 1 - 3 ) DM ( # 1 - 8 ) platelets . on HC ( n = 5 ) DM ( n = 18 ) individuals . ( * * p = 0 . 0025 vs . HC ) . GAPDH served as loading control .

B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.


C immunostaining for phosphorylated p53 in HC DM platelets . Graph indicates HC DM platelets positive for phosphorylated p53 signal . y - axis indicates of phosphorylated p53 positive from total ( * * p = 0 . 0064 vs . HC ) . Representative figure from n = 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.


D Mitochondrial potential ( ΔΨm ) was measured by MitoTracker ( 50nM for 15 min ) in HC DM platelets . Graph indicates signal of MitoTracker in HC DM platelets . y - axis indicates fold of signal in HC DM platelet ( * * p = 1 . 1608E - 05 vs HC ) . Signal of each group was converted to fold change compared with HC values . Representative figure from HC ( n = 6 ) DM ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).


E ΔΨm platelet apoptosis were measured by flow cytometry . ΔΨm was detected using TMRM apoptosis was assessed with Annexin - V ( PS externalization ) . Representative figure from n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.


F Western blot of apoptosis - related pp53 , p53 , cytochrome c , active caspase 3 in HC DM platelets . Tubulin was used as loading control . Representative figure from n = 3 .

F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.


G Western blot showing release of cytochrome c ( Cyto c ) into cytosol in DM . Representative figure from n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O

G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.


H EM of platelets mitochondria from HC DM demonstrating examples of mitochondria damage in DM platelets ( DM ) compared to a typical healthy mitochondria ( HC ) . Arrows indicate disrupted outer membranes . I of mitochondria in HC ( n = 33 ) DM ( n = 17 ) platelets . y - axis indicates average number of mitochondria in HC DM single platelet EM view ( * * p = 5 . 50811E - 05 vs . HC ) . J of healthy damaged mitochondria in HC DM platelets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.


A Western blot of autophagy markers , Beclin1 , ATG3 , ATG7 , ATG12 - 5 , LC3I / II , mitophagy - related Parkin PINK1 in HC ( # 1 - 3 ) DM platelets ( # 1 - 8 ) . B of autophagy markers mitophagy - related from HC ( # 1 - 5 ) DM ( # 1 - 18 ) individuals . ( Beclin1 ; * * p = 0 . 0003 , ATG12 ; * p = 0 . 0388 , ATG3 ; * p = 0 . 0213 , ATG7 ; p = 0 . 4051 , LC3 ; * p = 0 . 0298 , PINK1 ; * p = 0 . 0155 , Parkin ; * p = 0 . 0103 vs . HC ) . GAPDH served as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.


C three panels represent autophagy stages found in DM platelets . Autophagosome structures containing / enclosing organelles are indicated by black box . Shown for each panel is an enlargement of autophagosome ( DM1 2 ) or autolysosome ( DM3 ) . Dashed structures are an outline of autophagosome membranes .

C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.


D LC3 immuno - EM of HC DM platelets . Arrows indicate immunogold - labeled LC3 clusters . No clusters were found in HC platelets . Representative areas of clusters of gold labeling in DM ( DM1 - 3 ) are presented . Shown in insets are enlargements of gold clusters adjacent to mitochondria - like structures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.


E Double for MitoTracker ( mitochondria potential ) ( 50nM for 15 min ) LC3 ( autophagy ) in HC DM platelets . Boxed platelets are enlarged in Panel F . F A typical HC platelet has high potential low LC3 in contrast to a representative DM platelet which has low potential high LC3 . G of autophagosome positive platelets using 6 HC 4 DM samples after using CoxIV ( mitochondria ) LC3 ( autophagy marker ) . y - axis indicates of LC3 positive in total ( * * p = 0 . 006 vs . HC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).


A Representative western blot of recognized autophagy signaling molecules ( pJNK , JNK , pAKT , AKT , pmTOR , mTOR ) in HC ( # 1 - 3 ) DM ( # 1 - 8 ) platelets . of autophagy upstream signaling molecules on HC ( # 1 - 5 ) DM ( # 1 - 18 ) individuals . ( pAKT / AKT ; p = 0 . 0747 , pJNK / JNK ; * p = 0 . 001 , pmTOR / mTOR ; p = 0 . 3356 vs . HC ) . GAPDH was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.


B Representative western blot using HC HG platelets with or without H2O2 . assessing for LC3I / II . GAPDH was used as loading control .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.


C Platelet suspensions were incubated in HG for 2 hours with H2O2 ( 1mM ) . ROS were evaluated using a detection kit ( Enzo Life Science ) for 60 mins at 37 ºC .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.


D Western blot of p53 , JNK LC3I / II in HC platelets treated with H2O2 ( 1mM for 1 hour ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as loading control .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.


E Confocal microscopy was used to corroborate Western using triple for Mitotracker , CoxIV , LC3 in HC or H2O2 . insets highlight association of low potential with high of LC3 increased colocalization of Cox IV with LC3 in H2O2 - treated platelets .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.


F HC platelets were pretreated with H2O2 to induce autophagy then treated with or without SP600125 ( JNK inhibitor ) at 3 concentrations ( 1 , 5 10 µM ) to assess for inhibition of autophagy . This Western blot is representative of three independent experiments . of three independent experiments is provided at right . ( pJNK / JNK in H2O2 / SP600125 ; * * p = 0 . 0286 vs . H2O2 group , LC3II in H2O2 ; * p = 0 . 0241 vs . HC group , LC3II in H2O2 / SP600125 ; * * p = 0 . 0044 vs . H2O2 group , n = 3 for each group ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).


G Triple of CoxIV , LC3 , LAMP1 in HC H2O2 with / without SP600125 - treated platelets using confocal microscopy . Arrow indicates localization of LC3 LAMP1 . Graph indicates colocalization between LC3 , LAMP1 CoxIV signal . y - axis indicates fold change of colocalization between LC3 LAMP1 in mitochondria . Signal of each group was converted to fold compared with HC values ( H2O2 ; * p = 0 . 023 vs . HC , H2O2 / SP600125 ; * p = 0 . 04 vs . H2O2 group ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).


A Western blot of LC3 apoptosis markers phosphorylated p53 , p53 , LAMP1 in HC platelets . Platelets were treated with high glucose ( 25mM ) , with or without CCCP ( 10µM ) . This Western blot is representative of three independent experiments . B of phosphorylated p53 LAMP1 ( HG ; * p = 0 . 018 vs DMSO group , HG / CCCP ; * * p = 0 . 001 vs HG group , n = 4 for each group ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).


C recognized autophagy inhibitor ( 2mM 3 - Methyadenine ; 3MA for 1h ) was used to treat DM platelets compared to HC assessing for phosphorylated p53 , p53 , LC3I / IID of phosphorylated p53 / p53 was performed . Each value indicated average band density from a total of three independent samples ( DM / 3MA ; * p = 0 . 026 vs . DM group ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).


E Western blot of PLPs transfected with control or ATG3 siRNA , with or without treatment of 1 mM H2O2 . Each lane displayed a different transfected group . F of ATG3 , phosphorylated p53 ( ser15 ) , p53 , GAPDH in each group . ( ATG3 in ATG3 siRNA ; * * p = 0 . 0003 vs control group , pp53 in ATG3 siRNA / H2O2 ; * p = 0 . 0145 vs . H2O2 group ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).


A Western blot of phosphorylated p53 ( ser15 ) , p53 , LC3I / II , GAPDH in WT mouse platelets . Mouse platelets were treated high glucose ( 25mM ) with or without CCCP ( 10µM ) . B of phosphorylated p53 ( ser15 ) LC3 II ( pp53 in HG ; * * p = 0 . 009 vs WT group , pp53 in HG / CCCP ; * p = 0 . 022 vs HG group , LC3 II in HG / CCCP ; * p = 0 . 028 vs HG group , n = 3 for each group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).


C ΔΨm platelet apoptosis were measured by flow cytometry in WT or WT _ H2O2 or WT _ H2O2 / 3MA in mouse platelets . ΔΨm was detected using TMRM apoptosis were assessed with Annexin - V ( PS externalization ) . Representative of n = 3 . D Graph indicates of TMRM positive in total population ( H2O2 ; * * p = 0 . 002 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 008 vs . not treated group , NS means no significance , n = 3 ) . E Graph indicates of Annexin V positive in total population ( H2O2 ; NS , p = 0 . 058 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 004 vs . not treated group , * * p = 0 . 008 vs . H2O2 group , NS means no significance , n = 3 ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).


F Representative western blot of LC3 I / II in in WT or WT / H2O2 or WT / H2O2 / 3MA in mouse platelets ( n = 3 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).


G Representative western blot of phosphorylated p53 , Parkin in Parkin - / - mice compared to WT . of phosphorylated p53 Parkin ( Parkin ; * * p = 0 . 0001 vs . WT , pp53 ; * p = 0 . 019 vs . WT , n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Representative western blot analysis of phosphorylated p53, and Parkin in Parkin -/- mice compared to WT. Quantification analysis of phosphorylated p53 and Parkin (Parkin; **p=0.0001 vs. WT, pp53; *p=0.019 vs. WT, n=4).


H ΔΨm platelet apoptosis were measured by flow cytometry in WT or WT _ H2O2 Parkin - / - or Parkin - / - _ H2O2 mouse platelets . ΔΨm was detected using TMRM ( WT _ H2O2 ; * * p = 0 . 002 vs . WT group , Parkin - / - _ H2O2 ; * * p = 1 . 61525E - 06 vs . Parkin - / - group , n = 3 ) I Apoptosis level was assessed with Annexin - V ( PS externalization ) . Graph indicates of Annexin V positive in total population ( Parkin - / - : * p = 0 . 010 vs . WT group , Parkin - / - _ H2O2 ; * * p = 0 . 008 vs . WT _ H2O2 ; group , Parkin - / - _ H2O2 ; * * p = 0 . 001 vs . Parkin - / - group , NS means no significance n = 3 )

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)


J Western blot in WT Parkin - / - demonstrating inability to induce LC3 despite addition of H2O2 . Shown graphically are LC3II to LC3I ratios ( WT _ H2O2 ; * p = 0 . 028 vs . WT group , n = 3 )

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

J Western blot analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)


A Representative western blot of pp53 in WT ( n = 6 ) , DM ( n = 6 ) PINK1 - / - DM mice ( n = 3 ) . GAPDH served as loading control ( DM ; * * p = 0 . 006 vs . WT group , PINK1 - / - DM ; * * p = 0 . 004 vs . DM group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative western blot analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).


B Measurement of p - selectin translocation to by flow cytometry in WT ( n = 3 ) , DM ( n = 3 ) PINK1 - / - DM ( n = 3 ) miceplatelets . Graphs indicate mean ( MFI ) in each group . ( * p < 0 . 05 vs . WT or DM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Measurement of p-selectin translocation to the cell membrane by flow cytometry analysis in WT (n=3), DM (n=3) and PINK1 -/- DM (n=3) miceplatelets. Graphs indicate mean fluorescence intensity (MFI) in each group. (*p<0.05 vs. WT or DM).


C of vessel occlusion time after carotid arterial flow measurement ( WT DM ; PINK1 - / - : * * p < 0 . 01 vs . WT or DM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantification of vessel occlusion time after carotid arterial flow measurement (WT and DM; and PINK1 -/- : **p<0.01 vs. WT or DM).


D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury , indicative of occlusive thrombosis . Flow was plotted as of baseline flow before injury ( DM : n = 3 , PINK1 - / - DM ; n = 3 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).


A BAK - 6H permeabilising activity is exacerbated by cBID . BAK - 6H ( mBAK ∆ C21 - 6H ) was loaded at indicated concentrations onto Ni - NTA liposomes ( 5 μM ) prior to addition of cBID . release of carboxyfluorescein was measured over time . Results representative of at least 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.


B BAK - 6H oligomerises on liposomes induced by cBID . Liposomes were incubated with BAK - 6H ( 150 nM ) indicated of cBID or a BID BH3 peptide for 60 min . Liposomes were solubilised in digitonin BAK oligomers were analysed by BN - PAGE . In parallel , samples were tested at endpoint for liposome permeabilisation on release of flurorescent dextran , normalised to total of detergent permeabilised liposomes . Results representative of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.


C BAK - 6H oligomerisation on liposomes is blocked by BCL - XL . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( WT , 90 nM ) or cBID M97A ( 90 nM ) that has reduced affinity for BCL - XL , in presence or absence of BCL - XL for 60 mins prior to of BAK oligomerisation by BN - PAGE . Results representative of 2 independent experiments . Liposome permeabilisation was monitored with indicated combinations of cBID ( 90 nM ) , BAK - 6H ( 150 nM ) BCL - XL ( 1 μM ) . Data is presented as mean of 2 independent experiments with release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK cBid was approximately 50 % of detergent - lysis maximum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


D BAK - 6H oligomerisation on liposomes involves its BH3 domain . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( 90 nM ) . Antibody recognising BAK BH3 domain ( 4B5 ) or α1 - α2 loop ( 7D10 ) , as shown on structure of BAK ( PDB : 2IMS ) , were added ( 2 . 4 μg / μl ) either prior to ( pre ) or after ( post ) incubation with cBID for 60 mins . BAK : Ab is indicated ( arrowhead ) . 7D10 which recognises activated form of mBAK ( Iyer et al . , 2016 ) , bound to gel - shifted BAK when added either before or after activation with cBID . In contrast , 4B5 failed to appreciably gel - shift inactive BAK ( lanes 3 5 ) or BAK that was already activated ( lane 6 ) , but gel - shifted BAK when present during cBID activation ( lane 4 ) . Liposome permeabilisation was assessed after 20 min incubation with cBID ( 90 nM ) , BAK - 6H ( 150 nM ) antibodies 4B5 ( 2 . 5 μg ) or 7D10 ( 2 . 5 μg ) . Data is presented as mean of 2 independent experiments with release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK cBid was approximately 50 % of detergent - lysis maximum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


B BAK , or BAK with cysteine introduced at indicated positions ( on a BAK ∆ Cys background ) were stably expressed in Bax - / - Bak - / - MEFs assess for BAK expression by immunoblotting ( inset panel ; * indicates non - specific band . ) apoptotic activity in response to etoposide treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.


C E24 R169 are proximal in inactive BAK on mitochondria . Mitochondria - enriched fractions from were incubated with oxidant ( CuPhe ) induced intramolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


D E24 R169 dissociate during BAK activation . Mitochondria - enriched fractions from were incubated with cBID ( 100 nM ) prior to oxidant ( CuPhe ) induced intramolecular intermolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


C Bax - / - Bak - / - MEFs expressing indicated BAK variants were treated with doxycycline ( Dox , 3 h ) , incubated for a further 24 h in presence or absence of BH3 - mimetic compounds , was assessed by PI uptake .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.


D Mutation of BH4 promotes BAK activating conformation change . were treated with doxycycline ( 3 h ) to induce BAK expression followed by incubation with BH3 - mimetics where indicated ( for 2 h ) conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK ( G3172 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).


D BAK wild - type , or BAK with an engineered α1 - α2 loop thrombin cleavage site were stably expressed in Bax - / - Bak - / - MEFs . fractions were incubated with thrombin prior to incubation with copper phenanthroline ( CuPhe ) to induce disulphide - linkage . Samples were run on SDS - PAGE under non - reducing or reducing conditions immunoblotted for BAK with an antibody recognising BAK N - terminus upstream of thrombin cleavage site ( aa23 - 38 ) or an antibody recognising BH3 domain ( 4B5 ) downstream of cleavage site . Schematic indicates nature of BAK detected by each antibody . Disulphide - linkage indicated by a hashed . Note that N - terminal portion of thrombin BAK could not be detected with N - terminal antibody unless it was intramolecularly disulphide - linked . * cBID ( 100 nM ) was added to activate BAK during thrombin cleavage . Data representative of two independent experiments .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.


E Cleavage of BAK α1 - α2 loop potentiates MOMP . fractions from Bax - / - Bak - / - MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in presence or absence of cBID ( 10 nM ) for indicated times . ( P ) supernatant ( S ) fractions were separated immunoblotted for cytochrome c or BAK 4B5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.


( c ) Representative of a clustering with indicated on left . GFP mCherry are consistently displayed in cyan magenta ,

O O O O O O O O O O O O O O O O O O O O O

(c) Representative micrographs of a clustering assay with the proteins indicated on the left. GFP and mCherry fluorescence are consistently displayed in cyan and magenta, respectively


( a ) Top : schematic representation of STG - p62 construct . Bottom : characterization of several STG - p62 clones in comparison to parental Hap1

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Top: schematic representation of the STG-p62 construct. Bottom: characterization of several STG-p62 clones in comparison to parental Hap1 cells


( b ) Wild type or STG - p62 Hap1 were mock or puromycin treated for 2 hours stained for ubiquitin . endogenous p62 was stained with an anti - p62 antibody in parental Hap1 , while of GFP tag was recorded in STG - p62 . Arrowheads indicate co - localizing

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta


( c ) Purified STG - p62 was tested for cluster formation in presence of 20µM GST - 4xUb . Averages SDs of three independent replicates are shown for + GST - 4xUb sample of two for STG - p62

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Purified STG-p62 was tested for cluster formation in presence of 20µM GST-4xUb. Averages and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone


( d , e ) FCS of diffusion ( d ) brightness ( e ) of GFP1 - 5 ruler of STG - p62 in cytoplasm of Hap1 . Dashed lines represent linear regression of GFP1 - 5 ruler data . Regression coefficients are indicated . For data points averages SDs ( on both axes for STG - p62 ) are shown ( see methods for a list of number of measurements performed for each construct ) . MW : molecular . D : diffusion coefficient ( µm2 / s )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) FCS analysis of diffusion (d) and brightness (e) of the GFP1-5 ruler and of STG-p62 in the cytoplasm of Hap1 cells. Dashed lines represent the linear regression of the GFP1-5 ruler data. Regression coefficients are indicated. For all data points averages and SDs (on both axes for STG-p62) are shown (see methods for a list of the number of measurements performed for each construct). MW: molecular weight. D: diffusion coefficient (µm2/s)


( a ) Representative of cluster formation assays conducted with indicated

(a) Representative micrographs of cluster formation assays conducted with the indicated proteins


( c ) of cluster formation assays conducted with indicated . mCherry - p62 was pre - incubated with indicated 4xUb variants , followed by addition of streptavidin immediately before

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging


( d ) Representative of indicated samples taken 60 minutes after addition of

(d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin


( e ) of cluster formation assays conducted with indicated

(e) Quantification of cluster formation assays conducted with the indicated proteins


( f ) of cluster formation assays conducted with 2µM mCherry - p62 incubated with indicated of GST - GFP - 4xUb . Box : highest particle number per field detected in each sample was plotted against relative GST - GFP - 4xUb . Data points were fitted to a sigmoidal curve . regression coefficient is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Quantification of cluster formation assays conducted with 2µM mCherry-p62 incubated with the indicated concentration of GST-GFP-4xUb. Box: the highest particle number per field detected in each sample was plotted against the relative GST-GFP-4xUb concentration. Data points were fitted to a sigmoidal curve. The regression coefficient is indicated


( g ) of cluster formation assays conducted with GFP - p62 indicated GST - tagged ubiquitin

O O O O O O O O O O O O O O O O B-MEASURED_VAR

(g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains


( h - i ) of cluster formation assays performed with mCherry - p62 GST - 4xUb in presence of indicated free ubiquitin chains . Ubiquitin chains were either pre - mixed with p62 before addition of GST - 4xUb ( h ) , or added 3 minutes after addition of GST - 4xUb to p62 ( i )

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i)


( j ) of cluster formation assays conducted with mCherry - 2xFKBP - p62 in presence of AP20187 , GST - 4xUB indicated Ub

O O O O O O O O O O O O O O O O O O O O O O O O

(j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains


( k ) Representative of glutathione beads coated with indicated GST - tagged incubated with indicated mCherry - p62 variants . In pictures brightness was adjusted to highlight profile of beads . For graphs averages SDs from at least three independent replicates are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k) Representative micrographs of glutathione beads coated with the indicated GST-tagged proteins and incubated with the indicated mCherry-p62 variants. In all pictures the brightness was adjusted to highlight the profile of the beads. For all graphs averages and SDs from at least three independent replicates are shown


( a ) mCherry - p62 / GST - GFP - 4xUb clusters were photobleached recovery in bleached was monitored . mCherry - tagged low complexity domain ( LCD ) of hnRNPA1 ( hnRNPA1LCD ) was included as a reference for a liquid droplet - forming . Left : representative of bleached particles . arrows indicate position orientation of kymographs shown in Figure EV4a . Right : of recovery . traces of particles analyzed from three independent replicates are displayed

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) mCherry-p62/GST-GFP-4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored. The mCherry-tagged low complexity domain (LCD) of hnRNPA1 (hnRNPA1LCD) was included as a reference for a liquid droplet-forming protein. Left: representative micrographs of bleached particles. White arrows indicate the position and orientation of the kymographs shown in Figure EV4a. Right: quantification of fluorescence recovery. Individual traces of all particles analyzed from three independent replicates are displayed


( b ) p62 in STG - p62 were photobleached recovery was monitored . Left : representative of bleached . squares indicate bleached . Insert : magnification of bleached . Right : of recovery . Averages SDs of 28 ( unbleached ) 29 ( bleached ) from three independent experiments are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) p62 puncta in STG-p62 cells were photobleached and the fluorescence recovery was monitored. Left: representative micrographs of bleached cells. White squares indicate the bleached puncta. Insert: magnification of the bleached puncta. Right: quantification of fluorescence recovery. Averages and SDs of 28 (unbleached) and 29 (bleached) puncta from three independent experiments are shown


( c ) Representative electron of negatively stained p62 filaments incubated in presence or absence of GST - 4xUb for indicated

(c) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST-4xUb for the indicated times


( a ) Top : schematic representation of domain architecture of GFP - NBR1 construct . PB1 : Phox Bem1p domain , ZZ : Zinc finger , CC : coiled - coil , LIR : LC3 - interacting region , UBA : ubiquitin - associated domain . Bottom : GFP - NBR1 was recruited to glutathione beads coated with indicated GST fusion imaged by spinning disc microscopy . Representative of at least 70 beads imaged per sample from two independent experiments

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Top: schematic representation of the domain architecture of the GFP-NBR1 construct. PB1: Phox and Bem1p domain, ZZ: Zinc finger, CC: coiled-coil, LIR: LC3-interacting region, UBA: ubiquitin-associated domain. Bottom: GFP-NBR1 was recruited to glutathione beads coated with the indicated GST fusion proteins and imaged by spinning disc microscopy. Representative micrographs of at least 70 beads imaged per sample from two independent experiments


( b - c ) mCherry - p62 variants were recruited to GFP - or GFP - NBR1 - coated beads imaged as in ( a ) ( b ) Representative of at least 70 beads per sample from three independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b-c) mCherry-p62 variants were recruited to GFP- or GFP-NBR1-coated beads and imaged as in (a) (b) Representative micrographs of at least 70 beads per sample from three independent replicates