( C ) , Effect of p38MAPK inhibition on activation of AP - 1 family members in neutrophils stimulated with live GBS . Neutrophils were pretreated with SB202190 ( 5μM ) , stimulated for 90min with live GBS ( MOI 5 ) , lysed assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 , as determined by unpaired t - .

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(C), Effect of p38MAPK inhibition on the activation of AP-1 family members in neutrophils stimulated with live GBS. Neutrophils were pretreated with SB202190 (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of absence of Fpr1 , Fpr2 or TLR adaptor MyD88 on activation of AP - 1 family members in neutrophils . Neutrophils from WT , Fpr - or MyD88 - deficient mice were stimulated for 90 min with live GBS ( MOI 5 ) , lysed assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 vs WT mice , as determined by unpaired t - .

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(D), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on the activation of AP-1 family members in neutrophils. Neutrophils from WT, Fpr- or MyD88-deficient mice were stimulated for 90 min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001 vs WT mice, as determined by unpaired t-test.


( E ) , Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) .

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(E), Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS (MOI 5).


( F ) , Effect of absence of Fpr1 , Fpr2 or TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) . Shown is a blot representative of three separate experiments .

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(F), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS (MOI 5). Shown is a blot representative of three separate experiments.


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A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).


B , C ) ( B ) Colocalization of PD - L1 GFAP in AD ( C ) in control ( MX - O4 = amyloid deposits , upper picture : bar = 100 µm , lower picture : bar = 30 µm ) . D ) Immunohistochemical detection of PD - L1 in 9 - month - old APP / PS1 mice ( bar = 50 µM ) . E ) Immunohistochemical detection of PD - L1 in 4 - month - old APP / PS1 mice ( bar = 20 µm ) .

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B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).


A ) C6 expressing PD - L1 - myc treated with γ - secretase inhibitor DAPT for 18 h . PD - L1 was detected using a myc - antibody .

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A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.


B ) Immunoblot of HEK293 expressing PD - L1 - myc presenilin 1 ( hPS1 ) or a presenilin 1 dominant negative mutant ( hPS1 D246A ) .

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B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).


D ) 18 h 1 μM PMA treatment of C6 expressing PD - L1 - myc . Secretion of PD - L1 in conditioned medium ( CM ) was detected using antibody AF1019 .

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D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.


E ) Mouse astrocytes were incubated for 48 h with 1 μM PMA 10 μg / ml TNFα / 100 U / μl IFNγ lysates CM were immunoblotted using antibody AF1019 .

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E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.


A ) Colocalization of PD - 1 CD11b ( MX - 04 = methoxy - XO4 , stains amyloid deposits ) in AD by immunohistochemistry ( bar = 50 µm ) . B ) Colocalization of PD - 1 Iba1 or PD - 1 Aβ in a APP / PS1 mouse at 9 months of by immunohistochemistry ( bar = 20 µm ) .

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A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm).


E ) ELISA of TNFα secretion by wild type ( wt ) PD - 1 - / - microglia induced by 0 . 5 µM Aβ1 - 42 for 6 h in vitro ( n = 3 biological replicates , one - way ANOVA ( DF = 1 , F = 8 . 29 , p = 0 . 021 ) , Tukey ' s HSD , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) whiskers define + / - 1 . 5xIQR .

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E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


F - H ) Microglia from methoxy - XO4 - injected , 8 - month - old , wild type ( wt ) APP / PS1 mice were analyzed ( F ) for PD - 1 ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 13 . 99 , p = 0 . 0013 ) , Tukey ' s HSD , * * p < 0 . 01 ) , ( G ) for CD11b ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 30 . 06 , p = 5 . 47x10 - 05 ) , Tukey ' s HSD , * * * p < 0 . 001 ) ( H ) CD45 by flow cytometry ( n = 5 biological replicates per group , one - way ANOVA ( DF = 1 , F = 24 . 96 , p = 0 . 00013 ) , Tukey ' s HSD , * * * p < 0 . 001 ) normalized to expression of wild type mice ( represented by dashed lines ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) whiskers define + / - 1 . 5xIQR .

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F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


A , B ) ELISA of ( A ) RIPA soluble fraction of APP / PS1 APP / PS1 PD - 1 - / - mice for Aβx - 40 ( biological replicates with n = 6 for APP / PS1 n = 5 for APP / PS1 PD - 1 - / - , mean + / - , one - way ANOVA ( DF = 1 , F = 0 . 6 , p = 0 . 45 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) ( B ) of SDS soluble fraction ( biological replicates with n = 6 for APP / PS1 n = 5 for APP / PS1 PD - 1 - / - , mean + / - , one - way ANOVA ( DF = 1 , F = 32 . 8 , p = 2x10 - 5 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).


D , E ) Mice were analyzed for ( D ) of covered areas ( 5 sections per mouse analyzed with mean representing an data point ; biological replicates with n = 6 for APP / PS1 n = 9 for APP / PS1 PD - 1 - / - , mean + / - , one - way ANOVA ( DF = 1 , F = 0 . 905 , p = 0 . 90 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) ( E ) number of plaques per mm2 ( 5 sections per mouse analyzed with mean representing an data point ; biological replicates with n = 6 for APP / PS1 n = 9 for APP / PS1 PD - 1 - / - , mean + / - , one - way ANOVA ( DF = 1 , F = 4 . 08 , p = 0 . 054 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).


G ) Time needed to reach hidden platform ( latency in seconds ) in Morris water maze ( mean + / - of biological replicates with n = 12 for wt , n = 5 for PD - 1 - / - , n = 7 for APP / PS1 , n = 8 for APP / PS1 PD - 1 - / - , one - way ANOVA ( DF = 3 , F = 17 . 72 , p = 1x10 - 9 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) .

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G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).


A ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type or PD - 1 - / - microglia for up to 6 h ( mean + / - of a technical quadruplicate , Student ' s t - , * * p < 0 . 01 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) . B ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type microglia cultured in astrocyte - conditioned medium ( ACM ) from wild type or PD - L1 - / - astroglial cultures ( mean + / - of a technical quadruplicate , Student ' s t - , * p < 0 . 05 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate )

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A) In vitro phagocytosis of FAM-Aβ1-42 by wild type or PD-1-/- microglia for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis of FAM-Aβ1-42 by wild type microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate)


D ) of microglial in vivo phagocytosis in APP / PS1 APP / PS1 PD - 1 - / - mice at 8 . 5 month of after intraperitoneal injection of methoxy - XO4 ( biological replicates with n = 5 for APP / PS1 n = 4 for APP / PS1 PD - 1 - / - mice , Student ' s t - ( t = 2 . 1808 , df = 6 . 9829 ) , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) whiskers define + / - 1 . 5xIQR .

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D) Quantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


E - G ) Relative expression of surface marker ( E ) CD36 ( biological replicates with n = 5 for APP / PS1 n = 4 for APP / PS1 PD - 1 - / - mice , ANOVA ( DF = 1 , F = 0 . 002 , p = 0 . 94 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) , ( F ) CD11b ( biological replicates with n = 5 for APP / PS1 n = 4 for APP / PS1 PD - 1 - / - mice ) ( G ) CD45 on methoxy - XO4 + versus methoxy - XO4 - microglia in APP / PS1 APP / PS1 PD - 1 - / - mice normalized to methoxy - X04 - negative from APP / PS1 mice ( biological replicates with n = 5 for APP / PS1 n = 4 for APP / PS1 PD - 1 - / - mice ) Data information : central bands represent median , boxes show interquartile range ( IQR ) whiskers define + / - 1 . 5xIQR .

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E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


B ) Different expression of inflammasome - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia . C ) Different expression of complement system - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia .

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B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.


( a - d ) ELISA for IL - 1β ( a - c ) , IL - 18 ( d ) IL - 6 ( e ) in supernatants of resting or LPS - primed bone marrow - derived macrophages ( BMMs ) stimulated with palmitate conjugated to BSA ( PA - BSA ) or BSA control .

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(a-d) ELISA for IL-1β (a-c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control.


( e , f ) ELISA for IL - 1β ( e ) IL - 18 ( f ) in supernatants . Resting or LPS - primed BMMs generated from wild - type ( WT ) , Nlrp3 − / − , Pycard − / − or Nlrc4 − / − mice were stimulated with PA - BSA .

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(e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA.


( g ) IL - 1β ELISA of supernatants from LPS - primed BMMs stimulated with PA - BSA in absence or presence of pan - caspase inhibitor zVAD or caspase - 1 inhibitor zYVAD .

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(g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD.


( h ) ELISA for IL - 1β in supernatants . Resting or LPS - primed BMMs generated from WT or Casp1 - / - mice were stimulated with PA - BSA , BSA control , ATP or nigericin .

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(h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1-/- mice were stimulated with PA-BSA, BSA control, ATP or nigericin.


( i ) ELISA for TNF in supernatants . Resting or LPS - primed BMMs generated from WT , Nlrp3 - / - , Pycard - / - or Casp1 - / - mice were stimulated with PA - BSA as indicated . Values are expressed as mean ± s . d . , results are representative of three independent experiments .

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(i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3-/-, Pycard-/- or Casp1-/- mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.


Immunoblotting for procaspase - 1 activated caspase - 1 ( p10 ) , pro - IL - 1β IL - 1β ( p17 ) in supernatants ( Sup ) lysates ( Lys ) from resting or LPS - primed BMMs generated from WT , Nlrp3 − / − , Pycard − / − ( a ) or Nlrc4 − / − ( b ) mice stimulated with PA - BSA ( 0 . 2 or 0 . 5 mM ) as indicated at top . Data are representative of two independent experiments .

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Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.


( a ) ROS production determined by flow cytometry using fluoroprobe dihydrorhodamine 123 ( DHR ) in LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in absence or presence of ROS inhibitor APDC ( 50 μM ) . MFI , mean .

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(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity.


( b ) ELISA for IL - 1β on LPS - primed BMMs stimulated with PA - BSA in absence of or presence of increasing concentrations of APDC .

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(b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( c ) immunoblotting for caspase - 1 p10 IL - 1β p17 on LPS - primed BMMs stimulated with PA - BSA in absence of or presence of increasing concentrations of APDC .

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(c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( d ) ELISA for IL - 1β . Resting or LPS - primed BMMs were stimulated with PA - BSA ( 0 . 5 mM ) in absence or presence of ROS inhibitors APDC ( 10 or 50 μM ) N - acetyl - cysteine ( NAC , 5 or 25 mM ) or NAPDH oxidase inhibitor diphenyleneiodonium ( DPI , 5 or 25 μM ) . Values are expressed as mean ± s . d . In a , one of two independent experiments is shown . In each of b - d , results are representative of three independent experiments .

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(d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.


( a ) Thr172 - phosphorylated ( p - ) total AMPK - α subunit determined by immunoblotting . Resting or LPS - primed BMMs were untreated or stimulated with PA - BSA ( 0 . 5 mM ) for 24 h .

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(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h.


( b - e ) of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in absence or presence of AMPK agonist AICAR . ( b ) ROS production determined using fluoroprobe DHR .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR.


( b - e ) of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in absence or presence of AMPK agonist AICAR . ( c , d ) IL - 1β ( c ) IL - 6 ( d ) in supernatants , measured by ELISA .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA.


( b - e ) of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in absence or presence of AMPK agonist AICAR . ( e ) Immunoblotting for caspase - 1 , IL - 1β AMPK signaling molecules .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules.


( f ) Transfection of BMMs with empty vector ( EV ) or constitutively active ( CA ) AMPK - α1 subunit . N , non - transfected . Efficiency was determined 16 h later by flow cytometric of GFP expression in macrophages cotransfected with CA AMPK - α1 pmaxGFP . Values are percentages of GFP - positive in ranges indicated by bars . ( g ) ELISA for IL - 1β in supernatants of transfected as in f stimulated with PA - BSA ( 0 . 5 mM ) for 24 h . In each of a , b f , one of two independent experiments is shown . In each of c - e g , results are representative of three independent experiments . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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(f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a ) Immunoblotting for AMPK activation ( Thr172 p - AMPK - α ) LC3B ( top ) , densitometric to quantify of LC3B - II to actin ( bottom ) . BMMs were pretreated with LPS ( 200 ng / ml ) for 3 h , followed by PA - BSA ( 0 . 5 mM ) treatment for 24 h in absence or presence of chloroquine ( 50 μM ) . Med , medium control .

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(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control.


( b , c ) LC3B autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in absence or presence of AICAR . were fixed stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B in 100 ( b , right ) .

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(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right).


( b , c ) LC3B autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in absence or presence of AICAR . were fixed stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B in 100 ( b , right ) . were fixed examined by transmission electron microscopy for autophagosomes ( c ) . Quantitation ( c , right ) is on counting autophagosomes in ten per treatment .

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(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (c). Quantitation (c, right) is based on counting autophagosomes in ten cells per treatment.


( d ) Immunoblotting ( left ) densitometric to quantify of LC3B - II to actin ULK1 species ( right ) in treated BMMs .

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(d) Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs.


( e ) Mitochondrial ROS production determined using MitoSOX indicator . In each of a e , one of three independent experiments is shown . In each of b d , one of two independent experiments is shown . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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(e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a , b ) of FL83B mouse liver pretreated with mouse recombinant IL - 1β ( 2 ng / ml ) ( a ) or TNF ( 2 ng / ml ) ( b ) for 24 h , then stimulated with insulin ( 200 nM ) for 10 min . p - Akt ( Ser473 ) was determined by flow cytometry ( right ) with shown in graph at left ( a ) . p - Akt ( Ser473 ) was determined by immunoblotting ( b ) .

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(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).


( c ) p - IRS1 ( Ser307 ) , determined by immunoblotting , in FL83B mouse liver treated with either IL - 1β or TNF for 24 h .

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(c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B ( d , e ) or WT hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without stimulation .

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(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B ( d , e ) or WT hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without

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(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation


( g ) Akt serine 473 phosphorylation measured by flow cytometry . FL83B were pretreated with WT or Pycard − / − CM in absence or presence of IL - 1 receptor antagonist anakinra ( 1 μg / ml ) for 24 h , then stimulated with insulin .

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(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.


( h ) p - Akt ( Ser473 ) analyzed by immunoblotting quantified by densitometric in FL83B pretreated with WT CM in absence or presence of either anakinra or neutralizing antibody to TNF . results shown are representative of three independent experiments are expressed as mean ± s . d .

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(h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.


( a ) ITT performed 2 h after IL - 1β injection . Eight - week old C57BL / 6 mice were intraperitoneally injected with saline or mouse recombinant IL - 1β ( 1 μg / kg ) .

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(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg).


( b ) ITT in WT mice on regular diet ( RD ) or HFD for 12 , or Il1b - / - mice on HFD for 12 .

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(b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b-/- mice on HFD for 12 weeks.


( c , d ) ITT ( c ) serum IL - 1β ( d , left ) TNF ( d , right ) determined by ELISA , 2 h after IL - 1β injection . Recombinant mouse IL - 1β was administered to WT Tnfa − / − mice ( n = 5 for each group ) . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


( a ) Blood glucose insulin measured in WT ( n = 6 ) , Nlrp3 − / − ( n = 5 ) or Pycard − / − ( n = 6 ) mice under fasting or refed conditions after 12 of HFD .

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(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.


( b - e ) Glucose tolerance ( GTT ) ( b , d ) insulin tolerance ( ITT ) ( c , e ) performed for WT , Nlrp3 − / − ( b , c ) Pycard − / − ( d , e ) mice on HFD for 12 .

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(b-e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks.


( f ) GTT conducted on indicated bone marrow chimeric mice on HFD for 12 .

(f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks.


( g , h ) Insulin - stimulated phosphorylation of IRβ , IRS1 Akt ( Ser473 ) in liver tissues of WT Pycard − / − mice ( g ) , p - Akt ( Ser473 ) in liver , adipose ( WAT ) muscle tissues of WT Nlrp3 − / − mice ( h ) on HFD for 12 , after insulin ( 2 IU / kg ) infusion . Graphs at right of blots show quantitation of each molecule . pY , phosphotyrosine .

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(g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine.


( i , j ) Expression of Tnfa Mcp1 mRNA relative to Actb in liver tissues of Nlrp3 − / − mice ( i ) Pycard − / − mice ( j ) on regular diet ( RD ) or HFD for 12 . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


B . Phenotype of as indicated . ( a ) Sparse woolly hair . ( b ) Protrusion of upper lip due to deformity of teeth . ( c ) Ichthyosis - like fine scale erythema .

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B. Phenotype of the patients as indicated. (a) Sparse woolly hair. (b) Protrusion of upper lip due to deformity of teeth. (c) Ichthyosis-like fine scale and erythema.


C . of heart of as indicated . ( a ) M - mode echocardiography taken from parasternal long axis view of left ventricle ( LV ) illustrating poor LV function with severe involvement of interventricular septum ( IVS ) reduced motion of left ventricular posterior wall ( LVPW ) . ( b ) bi - dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function . SMOG is enhanced in right atrium . ( c ) bi - dimensional apical four chamber view with free tricuspid valve regurgitation ( TR ) due to lack of cooptation of tricuspid valve leaflets during systole ( marked by a red bar ) as a result of critical reduction of right ventricular function right ventricular enlargement . ( d ) Right left ventricular thrombi seen in short axis apical parasternal view . T1 - large right ventricular thrombus , T2 & amp ; amp ; T3 - left ventricular thrombi . ( e ) color - flow Doppler picture : TR . Free regurgitation of tricuspid valve shown in modified four chambers apical view . RA - right atrium , RV - right ventricle , LA - left atrium , LV - left ventricle , TR - tricuspid regurgitation color - flow Doppler .

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C. Images of the heart of the patients as indicated. (a) M-mode echocardiography taken from the parasternal long axis view of the left ventricle (LV) illustrating poor LV function with severe involvement of the interventricular septum (IVS) and reduced motion of the left ventricular posterior wall (LVPW). (b) bi-dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function. The SMOG is enhanced in the right atrium. (c) bi-dimensional apical four chamber view with free tricuspid valve regurgitation (TR) due to lack of cooptation of the tricuspid valve leaflets during systole (marked by a red bar) as a result of critical reduction of right ventricular function and right ventricular enlargement. (d) Right and left ventricular thrombi seen in the short axis apical parasternal view. T1-large right ventricular thrombus, T2&amp;T3-left ventricular thrombi. (e) The color-flow Doppler picture: TR. Free regurgitation of the tricuspid valve shown in modified four chambers apical view. RA-right atrium, RV-right ventricle, LA-left atrium, LV-left ventricle, TR-tricuspid regurgitation color-flow Doppler.


D . Photomicrografh demonstrating fibrosis interstitial inflammation in heart of VI10 . a c - post - mortem heart sections ( 2 . 7Y ) . ( a ) Prominent fibrosis lymphocytic interstitial inflammation ( Hematoxylin Eosin x50 ) . ( c ) Sub - epicardial inflammation mild interstitial " myocarditis " ( Hematoxylin Eosin x100 ) . ( b d ) - heart ( VI7 ) homozygous for p . Tyr747Ter . No inflammatory infiltrate or other histological abnormalities are seen . ( b ) Normal arrayed myocardium ( Hematoxylin Eosin x50 ) . ( d ) Sub - epicardial region with no histopathological changes . ( Hematoxylin Eosin x100 ) .

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D. Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10. a c- post-mortem heart sections (2.7Y). (a) Prominent fibrosis and lymphocytic interstitial inflammation (Hematoxylin Eosin x50). (c) Sub-epicardial inflammation and mild interstitial "myocarditis" (Hematoxylin Eosin x100). (b d)- Fetal heart (VI7) homozygous for p.Tyr747Ter. No inflammatory infiltrate or other histological abnormalities are seen. (b) Normal arrayed myocardium (Hematoxylin Eosin x50). (d) Sub-epicardial region with no histopathological changes. (Hematoxylin Eosin x100).


F . Segregation of c . 2241C > G , p . Y747X in PPP1R13L in studied family : affected girl VI10 , is homozygous for causative SV p . Y747X , as are four fetuses that were aborted . parents are obligate carriers as shown , of two healthy sisters , one carries wild allele only , other is heterozygous for causative SV .

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F. Segregation of c.2241C> G, p.Y747X in PPP1R13L in the studied family: The affected girl VI10, is homozygous for the causative SV p.Y747X, as are the four fetuses that were aborted. The parents are obligate carriers as shown and, of the two healthy sisters, one carries the wild allele only, and the other is heterozygous for the causative SV.


A . PPP1R13L mRNA expression level is lower in VI10 PDFs ( set as 1 ) than in CFs .

A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs.


B . C . Western blots ( WBs ) analyses demonstrated absence of iASPP in VI10 , VI4 VII1 PDFs .

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B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs.


D . WB demonstrated that PPP1R13L - shRNA downregulated putative iASPP band .

D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.


A . Higher induction of inflammatory cytokine mRNA in LPS - stimulated VI10 PDFs than in CFs . expression level in resting conditions of PDFs was set as 1 . Differences between knockdown control with p values ≤ 0 . 05 ( Student ' s t ) are indicated with an asterisk . B . as in A . for VII1 .

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A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1.


C . knockdown of PPP1R13L in CFs resulted in increased mRNA expression of inflammatory mediators . Differences between knockdown control with p values ( Student ' s t ) are indicated with asterisks in graph .

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C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks in the graph.


A . WB of fractions demonstrated that duration of p65 in nucleus is longer in VI10 PDFs than in CFs .

A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs.


B . ChIP shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS - stimulated PDFs than in LPS - stimulated - CFs . binding activity at IL1B promoter was set as 1 . Differences between knockdown control with p value = 0 . 00436 ( Student ' s t ) are indicated with an asterisk .

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B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk.


C . NF - kB inhibitor BOT - 64 blocked inducible mRNA expression of inflammatory cytokines . Differences between knockdown control with p values ( Student ' s t ) are indicated with an asterisks exact values .

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C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.


A . WB confirmed reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes .

A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes.


B . qPCR showed that ppp1r13l - knocked down murine cardiomyocytes ( using two alternative shRNA sequences ) expressed higher level of pro - inflammatory cytokine mRNAs after 4 hrs of LPS stimulation . expression in ppp1r13l - knocked down murine cardiomyocytes was set as 1 . Genome wide expression is presented in Figure 1S . Differences between knockdown control with p values ( Student ' s t ) are indicated with asterisks exact values .

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B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.


A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural regulatory genes as myosin binding C ( Mybpc3 ) , phospholamban ( Pln ) , ryanodine receptor 2 ( Ryr2 ) , sarcoglycan gamma ( Sgcg ) . Hearts removed from perfused mice mRNA was subjected for q PCR . Difference between knockdown control with p values = 0 . 03 ( Student ' s t ) is indicated with an asterisk .

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A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student's t test) is indicated with an asterisk.


B . A scatter plot showing deviation from normal expression by highlighting differentially expressed genes of newborn , 7 - week - 12 - week - hearts ( p < 0 . 05 threshold , calculated by Deseq algorithm " R " ) . expression level of s100a9 was higher in wa3 mice than in WT by 2 . 8 times . For full list of genes see Tables S1 S2 , functional analyses in Figures S3 - S6 .

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B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm "R"). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6.


C . Hierarchical clustering heat map of differentially expressed genes in hearts of 7 - 12 - week old wa3 mice in comparison to WT .

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C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.


A . LPS ( 5mg / Kg for 2 hrs ) induced higher expression of cytokine genes in wa3 hearts than in littermate WT hearts . results are presented as fold induction of untreated controls . mRNA expression of Pln Ryr2 was reduced in both wa3 WT hearts , while of Mybpc3 Sgcg was unchanged . For full list see Dataset EV3 for functional clustering in Appendix Figure S7 . Differences between knockdown control with p values = 0 . 04998 ( Student ' s t ) are indicated with an asterisk .

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A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk.


B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS ( 1mg / Kg ) .

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B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).


A - D SQST ‐ 1 : : GFP is very weakly expressed diffusely localized in cytoplasm in wild ‐ type larval intestine , but is expressed at much higher accumulates into numerous in intestine in bp399 mutant larvae . ( A ) ( C ) represent DIC of in ( B ) ( D ) , respectively .

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A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.


E Compared to wild ‐ type , SQST ‐ 1 : : GFP are much higher in bp399 bp412 mutant larvae as shown by immunoblotting . SQST ‐ 1 : : GFP in bp399 mutants are greatly reduced after 12 h of starvation . 200 young adult for each genotype were collected for .

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E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.


H , I SQST ‐ 1 : : GFP in rpl ‐ 43 ( bp399 ) mutants are separable from LAAT ‐ 1 : : Cherry ‐ labeled lysosomes under normal conditions , but are delivered to lysosomes upon starvation . Some are enclosed by lysosomes ( inserts ) . Scale bar : 20 μm .

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H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.


J - Q RNAi inactivation of let ‐ 363 , rpn ‐ 2 , cogc ‐ 1 , or hgrs ‐ 1 suppresses accumulation of SQST ‐ 1 : : GFP in rpl ‐ 43 ( bp399 ) mutants . ( J ) , ( L ) , ( N ) , ( P ) : DIC of in ( K ) , ( M ) , ( O ) , ( Q ) . Scale bar : 20 μm .

J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.


R SQST ‐ 1 : : GFP in rpl ‐ 43 ( bp399 ) mutants are greatly reduced by inactivation of rpt ‐ 3 rpn ‐ 2 . 200 young adult for each genotype were collected for .

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R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.


A , B SQST ‐ 1 : : GFP are absent in rpl ‐ 43 ( bp399 ) ; sma ‐ 3 ( wk20 ) mutant intestine .

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A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.


C of indicated with different of SQST ‐ 1 : : GFP . S : many SQST ‐ 1 : : GFP in intestinal , as in rpl ‐ 43 ( bp399 ) mutants ; M : fewer SQST ‐ 1 : : GFP in some but not intestinal ; N : no or very few SQST ‐ 1 : : GFP in intestinal . For each genotype , > 30 were examined .

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C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.


D SQST ‐ 1 : : GFP in rpl ‐ 43 ( bp399 ) mutants are dramatically decreased in a Western blot when sma ‐ 3 , lin ‐ 35 , or daf ‐ 2 is simultaneously depleted . Loss of function of daf ‐ 16 partially restores SQST ‐ 1 in rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutants .

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D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.


E Endogenous LGG ‐ 1 is elevated in a Western blot in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , sma ‐ 3 ( wk20 ) mutants .

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E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.


F mRNA of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , atg ‐ 7 are upregulated in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , sma ‐ 3 ( wk20 ) mutants . * * P 0 . 01 . 500 young adult were collected for . Error bars indicate s . d . from three experiments .

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F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


G , H No SQST ‐ 1 : : GFP are observed in lin ‐ 35 ( n745 ) ; rpl ‐ 43 ( bp399 ) mutant intestine .

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G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.


I , J Numerous SQST ‐ 1 : : GFP are formed in rpl ‐ 43 ( bp399 ) ; lin ‐ 15A ( n767 ) mutants .

I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.


K Endogenous LGG ‐ 1 are increased in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , lin ‐ 35 ( n745 ) mutants .

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K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.


L mRNA of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , atg ‐ 7 are upregulated in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , lin ‐ 35 ( n745 ) mutants . * * P 0 . 01 . 500 young adult were collected for . Error bars indicate s . d . from three experiments .

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


M , N rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutant larvae contain no intestinal SQST ‐ 1 : : GFP .

M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.


O , P SQST ‐ 1 : : GFP are partially restored in daf ‐ 16 ( mu86 ) ; rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) .

O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.


A , B hsp ‐ 4 : : GFP is very weakly expressed in wild ‐ type .

A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.


C of indicated with different of SQST ‐ 1 : : GFP . S : strong . M : medium . N : none . > 30 were examined for each genotype .

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C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.


D , E Phsp ‐ 4 : : GFP expression is much higher in rpt ‐ 3 ( RNAi ) .

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D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.


F , G Upregulation of Phsp ‐ 4 : : GFP expression in rpt ‐ 3 ( RNAi ) is suppressed by xbp ‐ 1 ( zc12 ) mutation .

F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.


H , I SQST ‐ 1 : : GFP are absent in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) .

H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.


J , K Loss of function of xbp ‐ 1 restores SQST ‐ 1 : : GFP in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) .

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J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.


M increase in bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , atg ‐ 7 mRNA in npl ‐ 4 . 1 , rpt ‐ 3 , pep ‐ 2 RNAi is dependent on xbp ‐ 1 . * P 0 . 05 , * * P 0 . 01 . 500 young adult were collected for . Error bars indicate s . d . from three experiments .

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M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


A Phsp ‐ 60 : : GFP is weakly expressed in wild ‐ type .

A Phsp‐60::GFP is weakly expressed in wild‐type animals.


B egl ‐ 46 ( RNAi ) causes upregulation of Phsp ‐ 60 : : GFP .

B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.


C of indicated mutant with different of SQST ‐ 1 : : GFP . S : strong . M : medium . N : none . > 30 were examined in each group .

C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.


D of SQST ‐ 1 : : GFP in various genetic mutants . 200 young adult for each genotype were collected for .

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D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.


E , F Loss of function of atfs ‐ 1 suppresses upregulation of Phsp ‐ 60 : : GFP in egl ‐ 46 ( n1126 ) mutants . ( E ) : DIC image of ( F ) .

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E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).


G , H dramatic decrease in number of SQST ‐ 1 : : GFP in intestine in rpl ‐ 43 ( bp399 ) ; egl ‐ 46 ( RNAi ) mutants ( G ) is restored by simultaneous loss of function of atfs ‐ 1 ( gk3094 ) ( H ) .

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G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).


I , J GFP : : LGG ‐ 1 forms numerous in intestine in egl ‐ 46 ( RNAi ) ( I ) , gfi ‐ 1 ( RNAi ) ( J ) .

I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.


( a ) Western blot of myosin - VI - depleted HeLa untreated or treated with 100 nM bafilomycin A1 . ( b ) of western blot LC3 - II ( ± s . d . ; n = 3 ) . * * P0 . 01 , * * * P0 .

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(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001


( c ) Confocal immunofluorescence microscopy of LC3 ( green ) in HeLa following knockdown of myosin VI . Hoechst labels nuclei ( blue ) . insets show higher magnifications of areas outlined in main ( d ) of LC3 - p62 - positive was performed results are represented as average per ( ± s . d . ; n = 3 ) from > 1 , 500 per experiment .

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(c) Confocal immunofluorescence microscopy of LC3 punctae (green) in HeLa cells following knockdown of myosin VI. Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images (d) Quantification of LC3- and p62-positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell (±s.d.; n = 3) from >1,500 cells per experiment.


( e ) Western blot of parental HeLa or HeLa stably expressing siRNA - resistant GFP - myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132 .

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(e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132.


( f ) Western blot of MEFs cultured from wild - type Snell ' s waltzer ( SV ) mice treated with 1 μM MG132 or 100 nM bafilomycin A1 .

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(f) Western blot analysis of MEFs cultured from wild-type and Snell's waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1.


( g ) of western blot LC3 - II ( ± s . d . ; n = 3 ) . * * P0 . 01 . ( h ) To evaluate effects on autophagosome biogenesis , results are shown as fold increase in normalized LC3 - II with bafilomycin A1 compared with untreated control . ( ± s . d . ; n = 3 ) .

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(g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3).


( i ) Western blot of cortical neurons from wild - type SV mice treated with 1 μM MG132 in absence or presence of 100 nM bafilomycin A1 . ( j ) of western blot LC3 - II was performed ( n = 2 ) . ( k ) To evaluate effects on autophagosome biogenesis , results are shown as fold increase in normalized LC3 - II with bafilomycin A1 compared with untreated control ( n = 2 ) .

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(i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2).


( l ) Western blot ( top ) confocal immunofluorescence microscopy ( bottom ) of mock - or myosin - VI - siRNA - treated HeLa stably expressing RFP - GFP - LC3 reporter . Hoechst labels nuclei ( blue ) . ( m ) Quantitative data of RFP GFP signal overlap from confocal of mock - or myosin - VI - siRNA - treated HeLa expressing RFP - GFP - LC3 . data are represented as Pearson ' s coefficient of RFP GFP signal correlation from > 100cells per experiment ( ± s . d . ; n = 3 ) .

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(l) Western blot (top) and confocal immunofluorescence microscopy (bottom) of mock- or myosin-VI-siRNA-treated HeLa cells stably expressing the RFP-GFP-LC3 reporter. Hoechst labels the nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock- or myosin-VI-siRNA-treated HeLa cells expressing RFP-GFP-LC3. The data are represented as the Pearson's coefficient of the RFP and GFP signal correlation from >100cells per experiment (±s.d.; n = 3).


n ) Confocal immunofluorescence microscopy of myosin - VI - depleted RPE stably expressing GFP - LC3 immunostained against GFP ( green ) cathepsin D ( red ) . Hoechst labels nuclei ( blue ) . insets show higher magnifications of areas outlined in main . Scale bars , 20 μm . Uncropped of blots are shown in Supplementary Fig . S9 .

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n) Confocal immunofluorescence microscopy of myosin-VI-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE transiently transfected with siRNA against myosin VI Atg5 were treated with 1 μM MG132 for 16 h . were processed for immunofluorescence microscopy following 16 h MG132 treatment ( zero time point ) 8 h post - washout of inhibitor . Immunolabelling for p62 was performed to visualize ( red ) Hoechst was used to identify nuclei ( blue ) .

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(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue).


( b ) Top , western blot confirming siRNA depletion . Bottom , of immunofluorescent p62 - positive was evaluated using an automated Cellomics VTi microscope . results were calculated as average p62 at 8 h post - MG132 washout normalized to zero time point are represented as of control ( ± s . d . ; n = 3 ) . * P0 . 05 , * * * P0 . 001 . See Methods Supplementary Table S1 for details of SMARTpool ( SP ) single ( 07 ) oligonucleotides .

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(b) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides.


( c ) Parental HeLa or HeLa stable expressing siRNA - resistant GFP - myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI were subsequently treated with 1 μM MG132 for 16 h . were processed for immunofluorescence microscopy at 16 h post - MG132 treatment ( t = 0 ) or allowed to recover following washout of MG132 for 8 h ( t = 8 ) . were processed for with automated Cellomics VTi microscope to evaluate p62 . results represent fold increase in p62 of myosin VI siRNA compared with mock control following recovery from MG132 washout ( t = 8 ; ± s . d . ; n = 3 ) .

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(c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP-myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3).


( d ) HeLa with stable expression of HttQ72 - GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction processing for immunofluorescence microscopy . Immunolabelling was performed for GFP ( green ) p62 ( red ) . Nuclei are labelled with Hoechst ( blue ) . ( e ) of HttQ72 - GFP was performed on myosin - VI - siRNA - transfected HeLa . results were calculated as of GFP - expressing with greater than 15 GFP - positive spots per . results represent mean ( ± s . d . ) from n = 3independent experiments , * * * P0 . 001 . insets show higher magnifications of areas outlined in main . Scale bars , 20 μm . Uncropped of blots are shown in Supplementary Fig . S9 .

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(d) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72-GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE transiently co - transfected with GFP - myosin VI cherry - LC3 either left untreated ( top row ) or treated with 250 nM Torin1 for 2 h ( bottom row ) were processed for immunofluorescence microscopy . arrows highlight areas of co - localization insets show higher magnifications of areas outlined in main .

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(a) RPE cells transiently co-transfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( b ) RPE treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI endogenous LC3 co - localization . arrows highlight areas of co - localization insets show higher magnifications of areas outlined in main .

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(b) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( c ) Image stills acquired from spinning - disc time - lapse video microscopy of RPE transiently co - transfected with cherry - LC3 GFP - myosin VI . outlined regions highlight areas of interest within that are shown to right ( i - iii ) as video image stills captured at 1 - min intervals . arrowheads highlight myosin - VI - positive vesicles ( green ) coming into contact co - localizing with LC3 - positive vesicles ( red ) .

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(c) Image stills acquired from spinning-disc time-lapse video microscopy of RPE cells transiently co-transfected with cherry-LC3 and GFP-myosin VI. The outlined regions highlight areas of interest within the cell that are shown to the right (i-iii) as video image stills captured at 1-min intervals. The arrowheads highlight myosin-VI-positive vesicles (green) coming into contact and co-localizing with LC3-positive vesicles (red).


( d ) Confocal immunofluorescence microscopy of RPE stably expressing cherry - LC3 following transient transfection with GFP - myosin VI . Immunostaining for GFP ( green ) cherry ( red ) was performed actin was visualized with phalloidin ( ) . Nuclei were labelled with Hoechst ( blue ) . arrowheads indicate areas of actin - rich myosin - VI / LC3 - positive autophagosomes . Scale bars , 20 μm ( a , b , d ) ; 10 μm ( c , whole - image ) ; 2 μm ( c , cropped ) .

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(d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).


( b ) RPE with stable expression of cherry - LC3 were transiently transfected with GFP - myosin VI cargo - binding tail domain containing various mutations in - interaction ( ΔWWY ΔRRL ) ubiquitin - binding motifs ( A1013G ) , followed by treatment with 250 nM Torin1 for 3 h to induce autophagy . Immunofluorescence microscopy was performed either in absence or presence of saponin extraction . arrowheads indicate areas of co - localization . Scale bar , 20 μm . ( c ) A Pearson ' s coefficient was calculated on basis of degree of co - localization between different GFP - myosin VI mutant tails cherry - LC3 from confocal immunofluorescence . graph represents data from more than 20 transfected from n = 2 independent experiments .

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(b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.


( a ) Left , immunofluorescence microscopy was performed on RPE following treatment with 100 nM bafilomycin A1 for 2 h . Immunolabelling was performed against indicated endogenous optineurin , T6BP NDP52 ( red ) , LC3 ( green ) . insets show higher magnifications of areas outlined in main . Right , from confocal , a Pearson ' s coefficient was calculated to estimate degree of co - localization of different autophagy adaptors with LC3 . As a negative control , a Pearson ' s coefficient was calculated after rotation of one colour image by 90 ° . results represent > 20 from n = 2 independent experiments .

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(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments.


( b ) Left , stable cherry - LC3 - expressing RPE were transiently transfected with GFP - myosin VI , treated with Torin1 for 3 h to induce autophagy , subsequently processed for confocal immunofluorescence microscopy . Areas of co - localization are in merged three - colour insets show higher magnifications of areas outlined in main . Right , a Pearson ' s coefficient was calculated for degree of co - localization between three colours . As a negative control , a Pearson ' s coefficient was calculated after rotating one colour image by 90 ° . Results represent > 10 from n = 2 independent experiments .

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(b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments.


( c ) HeLa were either mock transfected or co - transfected with siRNA targeted against TNO . were either left untreated or treated with100 nM bafilomycin A1 ( BfnA1 ) for 4 h . Western blot was performed against indicated . ( d ) of LC3 - II ( ± s . d . ) was performed from western blot data of TNO - siRNA treated HeLa . results represent n = 3independent experiments . * * P0 . 01 , * * * P0 .

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(c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001


( e ) Immunofluorescence microscopy of RFP - GFP - LC3 - expressing HeLa transfected with TNO siRNA was performed confocal were evaluated for correlation in GFP RFP signal overlap using Image JACoP plugin . results are calculated from > 100 from n = 2 independent experiments represented as Pearson ' s coefficient of GFP / RFP signal overlap . Scale bars , 20 μm . Uncropped of blots are shown in Supplementary Fig . S9 .

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(e) Immunofluorescence microscopy of RFP-GFP-LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson's coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Myosin VI Tom1 / Tom1L2 co - immunoprecipitate from LNCAP lysates . Immunoprecipitation of myosin VI with three separate affinity - purified rabbit polyclonal antibodies ( B4 , B7 , 2401 ) was performed alongside an IgG control immunoprecipitation . immunoprecipitates were analysed by western blotting with antibodies against Tom1 / Tom1L2 .

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(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2.


( b ) Binding of myosin VI to Tom1 requires WWY motif . Bottom , mammalian two - hybrid was used to binding of full - Tom1 Tom1L2 or various truncated versions of Tom1 , annotated by acid numbers , against wild - type myosin VI tail or myosin VI ΔWWY or ΔRRL tail . graph shows mean values from n = 2 independent experiments . Top , Tom1 domain structure truncation mutants . VHS , Vps - 27 , Hrs STAM ; GAT , GGA Tom1 .

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(b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1.


( c ) RPE stably expressing GFP - myosin VI were processed for immunofluorescence microscopy to evaluate GFP - myosin VI endogenous Tom1 / Tom1L2 or Rab5 co - localization . insets show higher magnifications of areas outlined in main panels . arrows indicate areas of co - localization .

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(c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization.


( d ) RPE with stable expression of GFP - myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot ( left ) immunofluorescence microscopy ( middle ) to evaluate GFP - myosin VI tail localization . were either fixed directly or saponin - extracted before fixation . ( right ) was performed on RPE stably expressing GFP - myosin VI tail transfected with mock , Tom1 or TNO siRNA . were processed for immunocytochemistry , immunolabelled for GFP nuclei labelled with Hoechst followed by of GFP - myosin VI tail per using an automated Cellomics VTi microscope . More than 600 per group from n = 3 independent experiments were analysed are represented as mean number of GFP - myosin VI tail per ( ± s . d . ) . Scale bars , 20 μm . Uncropped of blots are shown in Supplementary Fig . S9 .

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(d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) HeLa transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h . Western blot was performed on whole - lysates against indicated . ( b ) of LC3 - II of western blots was performed by an infrared imaging system . ( ± s . d . ; n = 3 ) . * P0 . 05 , * * P0 . 01 .

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(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01.


( c , left ) Confocal immunofluorescence microscopy was performed on HeLa transfected with Tom1 siRNA to evaluate LC3 formation ( green ) . insets show higher magnifications of areas outlined in main . Nuclei are labelled with Hoechst ( blue ) . ( c , right , d ) of LC3 - positive ( c , right ) p62 - positive ( d ) was performed in a 96 - well format on an Arrayscan VTi HCS microscope . were identified by Hoechst of nuclei ( blue ) . results are represented as average per ( ± s . d . ) from n = 3 independent experiments each performed in triplicate wells , each with > 500 .

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(c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells.


( e ) HeLa with stable expression of RFP - GFP - LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown ( left ) . Confocal immunofluorescence microscopy ( middle ) was performed were subsequently quantified for correlation between GFP RFP signals using ImageJ JACoP plugin ( right ) . results are for at least 100 from n = 3 independent experiments are represented as Pearson ' s coefficient of GFP / RFP overlap ( ± s . d . ) . A higher Pearson ' s coefficient of GFP / RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes .

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(e) HeLa cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown (left). Confocal immunofluorescence microscopy (middle) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin (right). The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson's coefficient of GFP/RFP overlap (±s.d.). A higher Pearson's coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes.


( f ) Confocal immunofluorescence microscopy of Tom1 - depleted RPE stably expressing GFP - LC3 immunostained against GFP ( green ) cathepsin D ( red ) . Hoechst labels nuclei ( blue ) . insets show higher magnifications of areas outlined in main .

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(f) Confocal immunofluorescence microscopy of Tom1-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images.


( g ) HeLa with stable expression of HttQ72 - GFP were transiently transfected with Tom1 siRNA ( top right ) , saponin - extracted , processed for immunofluorescence microscopy ( left ) to quantify HttQ72 - GFP ( bottom right ) . Immunolabelling against GFP ( green ) p62 ( red ) was performed . Nuclei ( blue ) were labelled with Hoechst . results are represented as number of GFP - expressing with greater than 15 GFP - positive spots per ( ± s . d . ; n = 3 ) . Scale bars , 20 μm . Uncropped of blots are shown in Supplementary Fig . S9 .

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(g) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72-GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Left , RPE with stable expression of GFP - LC3 following mock - or myosin - VI - siRNA transfection were processed for confocal immunofluorescence microscopy immunolabelled for Tom1 / Tom1L2 ( red ) . Nuclei ( blue ) were labelled with Hoechst . insets show higher magnifications of areas outlined in main panels arrows highlight areas of complete overlapping co - localization arrowheads highlight adjacent localization of Tom1 relative to LC3 . Right , was performed on > 75 for relative position ( adjacent or complete overlap ) of Tom1 - positive vesicles in relation to LC3 - positive vesicles . results are represented as average number of Tom1 - positive vesicles per categorized by their relative position to LC3 ( ± s . d . ; n = 3 ) .

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(a) Left, RPE cells with stable expression of GFP-LC3 following mock- or myosin-VI-siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labelled with Hoechst. The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co-localization and arrowheads highlight adjacent localization of Tom1 relative to LC3. Right, quantification was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. The results are represented as the average number of Tom1-positive vesicles per cell categorized by their relative position to LC3 (±s.d.; n = 3).