D MLKL - / - HT29 stably transfected with constructs encoding human MLKL - USP21 MLKL - USP21C221R , were treated with doxycycline , NSA ( 1 μM ) , TSI or combinations thereof ( added simultaneously ) . Sytox Green positive were quantified in real time by live imaging . Data are plotted as mean ± of six independent experiments . ( A red dashed is shown to highlight delay in kinetics upon treatment with NSA ) . E Human MLKL - USP21 MLKL - USP21C221R were inducibly expressed in MLKL - / - HT29 by doxycycline ( 10 ng / mL ) for 16 hrs , TSI was added for indicated times but samples were collected 25 hrs post - induction , followed by UBA - pulldown . Representative of three independent experiments .

O B-GENEPROD O O O B-CELL_LINE O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-SMALL_MOLECULE O O O B-ORGANISM B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O

D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.


( E ) Tadpoles at NF50 were amputated , fixed at indicated time then processed for whole mount in situ hybridisation using a probe specific for foxm1 . two last panels show transverse section in non - regenerating spinal cord ( nr ) regenerate at 3dpa . red circle highlights spinal cord asterisk notochord .

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(E) Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord.


( F ) Total was isolated from regenerating tails at indicated time points post amputation , reversed transcribed into cDNA analysed for foxm1 expression by qPCR , using ef1α as a reference gene . graph represents mean + / - SD of three independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons was used . * p = 0 .

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(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149


( A ) NF40 tadpoles with following genotypes foxm1 - / - ( mut ) , foxm1 + / - ( het ) foxm1 + / + ( wt ) were amputated left to regenerate for 7 days . show representative tails at 3 7 dpa . arrowheads indicate amputation site . ( B ) of rate of regeneration . of of regenerate to that has originally be amputated was compared for whole tail at 3 7 dpa . graph represents mean + / - SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment .

O O O O B-TISSUE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O 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-TISSUE 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-TISSUE O O O O

(A) NF40 tadpoles with the following genotypes foxm1-/- (mut), foxm1+/- (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site. (B) Quantification of the rate of regeneration. The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa. The graph represents the mean+/-SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment.


( D ) Representative of EdU ( red ) DAPI ( blue ) at 5 dpa . ( E ) graph represents mean + / - SD of EdU + over total number of in spinal cord of 5 to 12 tadpoles . Each data point represents a tadpole , with an average of 9 sections analysed per .

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(D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. (E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal.


( G ) Representative of EdU ( green ) , BrdU ( magenta ) , Sox3 ( ) DAPI ( blue ) at 3 dpa . ( H ) of in ( G ) . graph represents mean + / - SD of 6 tadpoles with an average of 13 sections per tadpole analysed .

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(G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. (H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed.


( C ) Representative sections labelled with an anti - PCNA antibody ( red ) DAPI ( blue ) at indicated day after amputation ( dpa ) . arrowheads point to in S phase in spinal cord yellow arrowhead at in G1 , G2 M phases . right panels correspond to inset indicated as a box in middle panels .

O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE 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 sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.


( A ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa fixed at 5 dpa . After sectioning , samples were labelled with antibodies against Sox3 ( green ) , EdU ( magenta ) DAPI ( blue ) . arrowheads show Sox3 positive extensions .

O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O

(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( B ) Sections of spinal cords at 5 dpa in stump ( non - regen . ) or in regenerate ( regen ) labelled with anti - Sox3 ( green ) , anti - Acetylated Tubulin ( AcTub , magenta ) DAPI ( blue ) . arrowheads show Sox3 positive extensions .

O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O

(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( E ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa fixed at 5 dpa . After sectioning , samples were labelled with antibodies against Myt1 ( green ) , EdU ( magenta ) DAPI ( blue ) . ( F ) of shown in ( E ) . graph represents mean + / - SD from 3 tadpoles with an average of 12 sections each .

O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O

(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.


( a ) microscopy of GFP - LC3 - transduced HeLa left unstimulated ( Unstim ) or stimulated for 4 h with C12 - iEDAP ( 2 . 5 μg / ml ) or for 2 h with rapamycin ( Rapa ; 50 μg / ml ) . Green , GFP - LC3 ; blue , DAPI ( - intercalating dye ) . Scale bar , 5 μm . ( b ) of dot - or ring - shaped GFP - LC3 signals ( representing autophagosomes ) in HeLa stimulated as described in a .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O

(a) Fluorescence microscopy of GFP-LC3-transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a.


( c ) Confocal microscopy of wild - type Nod2 - deficient BMDMs transduced with GFP - LC3 ( green ) left unstimulated or stimulated for 4 h with LPS ( 1 μg / ml ) or MDP ( 100 μg / ml ) or for 2 h with rapamycin ( 50 μg / ml ) . Scale bar , 15 μm . ( d ) of dot - or ring - shaped GFP - LC3 ( green ) in BMDMs stimulated as described in c .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O

(c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c.


( e ) Immunofluorescence microscopy of macrophages recovered from peritoneal cavities of thioglycolate - injected wild - type , Nod1 - Nod2 - deficient mice 4 h after intraperitoneal injection of MDP ( 300 μg / ml ) , FK565 ( 300 μg / ml ) or rapamycin ( 300 μg / ml ) ; after centrifugation by cytospin , were stained for endogenous LC3 ( red ) with DAPI ( blue ) . Scale bar , 10 μm . ( f ) of dot - or ring - shaped LC3 signals in macrophages recovered from peritoneal cavities of mice treated as described in c . Data are representative of four ( a - d ) or two ( e , f ) independent experiments with at least 100 per condition ( error bars ( b , d , f ) , s . d . ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a-d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).


( a ) Confocal microscopy of wild - type Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri M90T - RFP . ( b ) of colocalization of GFP - LC3 signals with in a .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

(a) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri M90T-RFP. (b) Quantification of the colocalization of GFP-LC3 signals with bacteria in a.


( c ) Confocal microscopy of wild - type Nod1 - deficient GFP - LC3 - expressing MEFs infected for 2 h with S . flexneri ΔIcsB - RFP . ( d ) of colocalization of GFP - LC3 signals with in c .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

(c) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3-expressing MEFs infected for 2 h with S. flexneri ΔIcsB-RFP. (d) Quantification of the colocalization of GFP-LC3 signals with bacteria in c.


( e , f ) Plate of in wild - type or Nod1 - deficient MEFs infected for 0 . 5 h with S . flexneri ΔIcsB in presence or absence of autophagy blocker LY294002 ( LY ) or protease inhibitors ( PI ) , followed by 5 . 5 h of incubation in presence of gentamicin .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O

(e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin.


( g - j ) Confocal microscopy ( g , i ) of colocalization of GFP - LC3 signals with M90T ΔIcsB ( h , j ) in wild - type RIP2 - deficient ( Ripk2 − / − ) GFP - LC3 - expressing MEFs infected for 2 h with wild - type S . flexneri ( M90T ; g , h ) or ΔIcsB S . flexneri ( i , j ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O

(g-j) Confocal microscopy (g,i) and quantification of the colocalization of GFP-LC3 signals with M90T and ΔIcsB (h,j) in wild-type and RIP2-deficient (Ripk2−/−) GFP-LC3-expressing MEFs infected for 2 h with wild-type S. flexneri (M90T; g,h) or ΔIcsB S. flexneri (i,j).


( k ) Enzyme - linked immunosorbent of KC secretion by wild - type RIP2 - deficient MEFs infected for 0 . 5 h with M90T incubated for 5 . 5 h in presence of gentamicin .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O

(k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin.


( l , m ) Confocal microscopy ( l ) of colocalization of GFP - LC3 signals ( green ) with M90T ΔIcsB ( m ) of wild - type NEMO - deficient ( Ikbkg − / − ) GFP - LC3 - expressing MEFs infected for 0 . 5 h with wild - type S . flexneri ( M90T ) or S . flexneri ΔIcsB incubated for 1 . 5 h in presence of gentamicin . Red , RFP . Arrows indicate colocalization of GFP - LC3 signals with S . flexneri . Scale bars , 10 μm . * P 0 . 05 , * * P 0 . 01 ( t - ) . Data represent three independent experiments with at least 100 each ( error bars , s . e . m . ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3-expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).


( a ) Immunofluorescence of HeLa expressing hemagglutinin - tagged Nod1 ( HA - Nod1 ( red ) ; top ) or HA - Nod2 ( red ; bottom ) . Blue , DAPI .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O

(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI.


( b ) Immunofluorescence of HeLa expressing Myc - tagged FL - ATG16L1 ( Myc - FL - ATG16L1 ) .

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(b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc-FL-ATG16L1).


( c , d ) Immunofluorescence of HeLa coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) Myc - FL - ATG16L1 ( green ) . Blue , DAPI .

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(c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc-FL-ATG16L1 (green). Blue, DAPI.


( e ) Immunofluorescence of HeLa expressing a Flag - tagged truncated form of ATG16L1 ( Flag - ΔN85 - ATG16L1 ; green ) . Blue , DAPI .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O

(e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag-ΔN85-ATG16L1; green). Blue, DAPI.


( f ) Immunofluorescence of HeLa coexpressing HA - Nod2 ( red ) Flag - ΔN85 - ATG16L1 ( green ) . Blue , DAPI .

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(f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag-ΔN85-ATG16L1 (green). Blue, DAPI.


( g ) Immunofluorescence microscopy of Mode - K stained for endogenous Nod2 ( green ) ATG16L1 ( red ) with DAPI ( blue ) . For a - g , arrows indicate colocalization of ATG16 Nod2 ; outlined at left is enlarged at right ( a , b , e ) or in other ( c , d , f , ) ; scale bars , 8 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O 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) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a-g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm.


( h , i ) Immunoprecipitation ( IP ) immunoblot ( IB ) of HEK293 transfected with Flag - ΔN85 - ATG16L1 ( h ) or Myc - FL - ATG16L1 ( i ) , together with HA - Nod1 , HA - Nod2 or HA - tagged enhanced GFP ( EGFP - HA ) . Ig , immunoglobulin ( loading control ) . Results are representative of one of at least three independent experiments ( a - g ) or one of three independent experiments ( h , i ) .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O 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) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag-ΔN85-ATG16L1 (h) or Myc-FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a-g) or one of three independent experiments (h,i).


Immunofluorescence microscopy of wild - type RIP2 - deficient MEFs expressing Flag - tagged ΔN85 - ATG16L1 HA - Nod1 ( a ) or HA - Nod2 ( b ) . Scale bars , 10 μm . outlined at far left is enlarged at right . Data are representative one of two independent experiments .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy of wild-type and RIP2-deficient MEFs expressing Flag-tagged ΔN85-ATG16L1 and HA-Nod1 (a) or HA-Nod2 (b). Scale bars, 10 μm. Area outlined at far left is enlarged at right. Data are representative one of two independent experiments.


( a ) microscopy of immortalized GFP - LC3 - transduced lymphoblasts from donors homozygous for normal ATG16L1 * 300T allele ( 300T ) or risk allele ATG16L1 * 300A ( 300A ) , left unstimulated or treated for 2 h with rapamycin ( 50 μg / ml ) or for 4 h with MDP ( 20 μg / ml ) . Green , GFP - LC3 . Scale bar , 5 μm . ( b ) Induction of GFP - LC3 signals by rapamycin , MDP or Gram - positive peptidoglycan ( PG + ; 20 μg / ml ) in treated as described in a , presented relative to GFP - LC3 signals in unstimulated control . * P 0 . 05 ( t - ) . Data represent three independent experiments ( error bars ( b ) , s . d . ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O 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) Fluorescence microscopy of immortalized GFP-LC3-transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).


( a ) Immunofluorescence microscopy of HeLa transfected with HA - Nod2 ( red ) Flag - ΔN85 - ATG16L1 ( green ) infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 ATG16L1 around invading . Scale bars , 5 μm . Data are representative of one of three independent experiments .

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(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag-ΔN85-ATG16L1(green) and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( b ) Immunofluorescence microscopy of endogenous Nod2 ATG16L1 in Mode - K infected for 10 - 20 min with S . flexneri M90T . Blue , DAPI . Arrows indicate colocalization of Nod2 ATG16L1 around invading . Scale bars , 5 μm . Data are representative of one of three independent experiments .

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(b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.


( a , b ) Immunofluorescence microscopy of HeLa expressing Flag - ΔN85 - ATG16L1 ( green ; a ) or Myc - FL - ATG16L1 ( green ; b ) HA - tagged Nod2 L1007fsinsC ( HA - Nod2 , 1007fs ) . outlined at left is enlarged in other .

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(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (green; a) or Myc-FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA-Nod2, 1007fs). Area outlined at left is enlarged in other images.


( c , d ) Immunofluorescence microscopy of HeLa expressing Flag - ΔN85 - ATG16L1 ( c ) or Myc - FL - ATG16L1 ( d ) together with HA - tagged Nod2fs ( red ) . Blue , DAPI . Scale bar , 8 μm .

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(c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm.


( e ) Immunofluorescence microscopy of wild - type Nod2fs - KI BMDMs transduced with GFP - LC3 ( green ) infected for 0 . 5 h with S . flexneri M90T - RFP ( red ) , followed by incubation for 1 h with gentamicin . Arrows indicate no colocalization of Nod2 with ΔN85 - ATG16L1 or FL - ATG16L1 . Scale bar , 15 μm . ( f ) of colocalization of GFP - LC3 signals with S . flexneri M90T - RFP in experiments as described in e . Data are representative of one of three independent experiments ( error bars ( f ) , s . d . ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O 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) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).


D ) heart , tibialis anterior ( TA ) triceps muscle were isolated analyzed to identify presence of duplication from exons 18 to 30 via RT - PCR using primers represented by arrows . Sanger sequencing confirmed correct splicing of exons 30 18 at duplication junction .

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D) The heart, tibialis anterior (TA) and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT-PCR using primers represented by the arrows. Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction.


A ) 8 - µm cross section of 15 - week - old WT Dup18 - 30 TA triceps were analyzed for dystrophin localization by immunofluorescence . Asterisk indicates clusters of revertant . Scale bars , 100 μm . B ) muscle architecture of same was investigated by H & E . A representative image is shown . Scale bars , 100 μm . Asterisk indicates areas with necrotic fibrosis . Arrows indicate with central nuclei .

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A) 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm. B) The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei.


G , H ) Mice were tested in an open - field chamber in which total travelled ( G ) vertical activity ( H ) were assessed . WT , n = 9 ; Dup18 - 30 n = 12 .

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G, H) Mice were tested in an open-field chamber in which total distance travelled (G) and vertical activity (H) were assessed. WT, n = 9; Dup18-30 n = 12.


F , G ) Western blotting detected restoration of dystrophin expression in ( F ) heart ( G ) TA in Dup18 - 30 mice . 25 % 50 % of WT compared to Dup18 - 30 mice have been loaded on gel . Calnexin was used as a loading control .

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F, G ) Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.


H ) Immunostaining showed restoration of dystrophin expression in heart TA . A representative sample is shown . Scale bars , 100 μm .

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H) Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm.


A ) Immunofluorescence for alpha - syntrophin beta - sarcoglycan in TA triceps of Dup18 - 30 mice . A representative image is shown . Scale bars , 100 μm .

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A) Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm.


B ) TA , triceps diaphragm muscle architecture was analyzed by H & E . Scale bars , 50 μm .

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B) TA, triceps and diaphragm muscle architecture was analyzed by H&E staining. Scale bars, 50 μm.


A ) Fibrosis was analyzed in diaphragm via Masson Trichrome . Scale bars , 100 μm .

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A) Fibrosis was analyzed in the diaphragm via Masson Trichrome staining. Scale bars, 100 μm.


WT , Dup18 - 30 untreated Dup18 - 30 treated mice were tested 7 after treatment by measuring ( C ) forelimbs grip

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WT, Dup18-30 untreated and Dup18-30 treated mice were tested 7 weeks after treatment by measuring (C) forelimbs grip strength


A HEK293T were transfected with NF - kBluciferase reporter plasmid FLAG - tagged full - CARD14 ( FL ) or CARD14sh ( short ) at indicated concentrations analyzed 24 h later . ( A ) NF - kB reporter gene expression .

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A HEK293T were transfected with NF-kBluciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (A) NF-kB reporter gene expression.


HEK293T were transfected with NF - kB luciferase reporter plasmid FLAG - tagged full - CARD14 ( FL ) or CARD14sh ( short ) at indicated concentrations analyzed 24 h later . ( B ) IL - 8 secretion . Values are mean of triplicates ± s . e . P - values are compared against empty vector treatment . Significance , ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 by student t . Unless otherwise stated , p - values are p < 0 . 001 .

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HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (B) IL-8 secretion. Values are the mean of triplicates ± s.e. P-values are compared against empty vector treatment. Significance levels, ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Unless otherwise stated, p-values are p<0.001.


HEK293T were transfected with NF - kB luciferase reporter plasmid FLAG - tagged full - CARD14 ( FL ) or CARD14sh ( short ) at indicated concentrations analyzed 24 h later . ( C ) Immunoblotting for c - Jun , phospho - c - Jun , JNK , phospho - JNK , p38 phospho - p38 as indicated . Actin was used as a loading control . Data are representative of two independent experiments .

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HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (C) Immunoblotting for c-Jun, phospho-c-Jun, JNK, phospho-JNK, p38 and phospho-p38 as indicated. Actin was used as a loading control. Data are representative of two independent experiments.


( A ) MALT1 interacts with CARD14sh in a BCL10 - dependent manner . HEK293T were transfected with FLAG - CARD14sh , Myc - MALT1 FLAG - BCL10 as indicated . 24 h later , MALT1 was immunoprecipitated ( IP ) from lysates with anti - MALT1 . CARD14sh BCL10 co - immunoprecipitation with MALT1 was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - myc . closed arrowhead shows position of CARD14 , double arrowhead shows position of BCL10 . asterisk indicates a non - specific band . Total expression of transfected are shown by immunoblotting of a fraction of lysates with indicated antibodies ( bottom panel ) .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(A) MALT1 interacts with CARD14sh in a BCL10-dependent manner. HEK293T cells were transfected with FLAG-CARD14sh, Myc-MALT1 and FLAG-BCL10 as indicated. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1. CARD14sh and BCL10 co-immunoprecipitation with MALT1 was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-myc. The closed arrowhead shows the position of CARD14, the double arrowhead shows the position of BCL10. The asterisk indicates a non-specific band. Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies (bottom panel).


( B ) MALT1 co - expression potentiates CARD14sh - induced JNK p38 MAP kinase activation . HEK293T were transfected with indicated concentrations of FLAG - tagged CARD14sh , with or without MALT1 ( 30ng ) . Transfection with FLAG - TRAF6 FLAG - CARD11 ( L232LI ) were used as a positive control . c - Jun accumulation , JNK p38 phosphorylation were analyzed by immunoblotting with antibodies indicated . Actin was used as a loading control .

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(B) MALT1 co-expression potentiates CARD14sh-induced JNK and p38 MAP kinase activation. HEK293T cells were transfected with the indicated concentrations of FLAG-tagged CARD14sh, with or without MALT1 (30ng). Transfection with FLAG-TRAF6 and FLAG-CARD11(L232LI) were used as a positive control. c-Jun accumulation, JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.


( C ) MALT1 co - expression potentiates NF - kB activation by pathogenic CARD14 mutants . HEK293T were transfected with NF - kB reporter plasmid wild - type ( WT ) FLAG - CARD14 or indicated psoriasis - associated CARD14 mutants ( 15ng ) , with or without MALT1 ( 15ng ) . Luciferase activity in lysates was measured after 24 h . Values are mean of triplicates ± s . e . Significance , * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 by student t . Expression of transfected was verified by western blotting ( bottom panel ) .

O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O

(C) MALT1 co-expression potentiates NF-kB activation by pathogenic CARD14 mutants. HEK293T were transfected with NF-kB reporter plasmid and wild-type (WT) FLAG-CARD14 or the indicated psoriasis-associated CARD14 mutants (15ng), with or without MALT1 (15ng). Luciferase activity in cell lysates was measured after 24 h. Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Expression of transfected proteins was verified by western blotting (bottom panel).


( D ) CARD14 ( E138A ) mutation enhances MALT1 binding . HEK293T were transfected with wild - type ( WT ) FLAG - CARD14sh or indicated CARD14sh mutants Myc - MALT1 . 24 h later , MALT1 was immunoprecipitated ( IP ) from lysates with anti - MALT1 CARD14 co - immunoprecipitation was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - MALT1 . asterisk indicates a non - specific band . Immunoprecipitation with a non - relevant antibody ( rIgG ) was used as a negative control ( first lane ) . Total expression of transfected are shown by immunoblotting of total lysates ( bottom panel ) . Data are representative of two independent experiments .

O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O

(D) CARD14(E138A) mutation enhances MALT1 binding. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14sh or the indicated CARD14sh mutants and Myc-MALT1. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1 and CARD14 co-immunoprecipitation was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-MALT1. The asterisk indicates a non-specific band. Immunoprecipitation with a non-relevant antibody (rIgG) was used as a negative control (first lane). Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates (bottom panel). Data are representative of two independent experiments.


( A ) MALT1 expression is essential for CARD14sh - induced NF - kB activation . MALT1 - deficient HEK293T were transfected with NF - kB reporter plasmid indicated expression plasmids . Luciferase activity in lysates was measured 24 h later . CARD11 ( L232LI ) TRAF6 transfection were used as MALT1 - dependent MALT1 - independent controls , respectively . Values are mean of triplicates ± s . e . Expression of transfected plasmids was verified by immunoblotting ( lower panel ) .

O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O

(A) MALT1 expression is essential for CARD14sh-induced NF-kB activation. MALT1-deficient HEK293T cells were transfected with NF-kB reporter plasmid and the indicated expression plasmids. Luciferase activity in cell lysates was measured 24 h later. CARD11(L232LI) and TRAF6 transfection were used as MALT1-dependent and MALT1-independent controls, respectively. Values are the mean of triplicates ± s.e. Expression of the transfected plasmids was verified by immunoblotting (lower panel).


( B ) MALT1 expression is essential for CARD14sh - induced JNK activation . MALT1 - deficient HEK293T were transfected with FLAG - CARD14sh ( 30ng ) either alone or together with either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) as indicated . c - Jun accumulation JNK phosphorylation were analyzed by immunoblotting with antibodies indicated . Actin was used as a loading control .

O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O

(B) MALT1 expression is essential for CARD14sh-induced JNK activation. MALT1-deficient HEK293T cells were transfected with FLAG-CARD14sh (30ng) either alone or together with either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A) as indicated. c-Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.


( C ) MALT1 deficiency inhibits CARD14 - induced gene expression in HaCaT keratinocytes . HaCaT were transfected with scrambled ( scr ) or MALT1 - targeting siRNA prior to doxycycline - induced CARD14 expression as described in materials methods . IL - 8 MCP - 1 in supernatants was measured 8 h ( IL - 8 ) or 24 h ( MCP - 1 ) later by ELISA . Values are mean of triplicates ± s . e . Doxycycline - induced CARD14 expression MALT1 knock - down were verified by immunoblotting ( right panel ) . Data are representative of two independent experiments .

O O O B-GENEPROD O O B-GENEPROD O O O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.


( A ) CARD14 signaling induces CYLD A20 cleavage . HEK293T were transfected with different concentrations of FLAG - CARD14 , with or without MALT1 as indicated . CARD11 ( L232LI ) transfection was used as a positive control . Endogenous CYLD A20 processing was analyzed by immunoblotting . CYLD A20 cleavage fragments are indicated by an arrow .

O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O

(A) CARD14 signaling induces CYLD and A20 cleavage. HEK293T cells were transfected with different concentrations of FLAG-CARD14, with or without MALT1 as indicated. CARD11(L232LI) transfection was used as a positive control. Endogenous CYLD and A20 processing was analyzed by immunoblotting. CYLD and A20 cleavage fragments are indicated by an arrow.


( B ) CARD14 signaling induces CYLD cleavage after R324 . HEK293T were transfected with different concentrations of FLAG - CARD14sh either wild - type ( CYLDWT ) or non - cleavable CYLD ( CYLDR324A ) . CYLD processing was analyzed by immunoblotting . CYLD cleavage fragment is indicated by an arrow .

O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

(B) CARD14 signaling induces CYLD cleavage after R324. HEK293T cells were transfected with different concentrations of FLAG-CARD14sh and either wild-type (CYLDWT) or non-cleavable CYLD (CYLDR324A). CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.


( C ) Expression of catalytically active MALT1 is necessary for CARD14 - induced CYLD cleavage . MALT1 - deficient HEK293T in which MALT1 expression was restored by transfection with different concentrations of either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) , were co - transfected with FLAG - CARD14sh CYLD as indicated . CYLD processing was analyzed by immunoblotting . CYLD cleavage fragment is indicated by an arrow .

O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

(C) Expression of catalytically active MALT1 is necessary for CARD14-induced CYLD cleavage. MALT1-deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A), were co-transfected with FLAG-CARD14sh and CYLD as indicated. CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.


( D ) Psoriasis - associated CARD14 mutation enhances activation of MALT1 proteolytic activity . HEK293T were transfected with wild - type ( WT ) FLAG - CARD14 or different psoriasis - associated CARD14 mutants ( 20ng ) , with or without MALT1 ( 20 ng ) as indicated . Endogenous CYLD A20 processing , as well as IκBα phosphorylation , were analyzed by immunoblotting . CYLD A20 cleavage fragments are indicated by an arrow .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O

(D) Psoriasis-associated CARD14 mutation enhances activation of MALT1 proteolytic activity. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14 or different psoriasis-associated CARD14 mutants (20ng), with or without MALT1 (20 ng) as indicated. Endogenous CYLD and A20 processing, as well as IκBα phosphorylation, were analyzed by immunoblotting. The CYLD and A20 cleavage fragments are indicated by an arrow.


( E ) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14 - induced gene expression in HaCaT keratinocytes . CARD14 expression was induced by doxycycline ( dox ) treatment in absence or presence of different concentrations of mepazine as indicated . next morning , culture medium was refreshed IL - 8 MCP - 1 in supernatant was measured 8 h later by ELISA . TNF stimulation ( MALT1 - independent ) was used as a negative control . Values are mean of triplicates ± s . e . Data are representative of two independent experiments . Significance , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


( A - B ) CYLD A20 inhibit CARD14sh - induced NF - kB activation . HEK293T were transfected with NF - kB reporter plasmid FLAG - tagged CARD14sh ( 30ng ) , with or without indicated concentrations of CYLD ( A ) or A20 ( B ) . Luciferase activity in lysates was measured 24 h later . Closed arrowheads indicate full A20 CYLD ; arrows indicate A20 CYLD . Data are representative of three independent experiments .

O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O

(A-B) CYLD and A20 inhibit CARD14sh-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid and FLAG-tagged CARD14sh (30ng), with or without the indicated concentrations of CYLD (A) or A20 (B). Luciferase activity in cell lysates was measured 24 h later. Closed arrowheads indicate full length A20 and CYLD; arrows indicate cleaved A20 and CYLD. Data are representative of three independent experiments.


( C ) A20 ABIN - 1 inhibit CARD14 - induced NF - kB activation . HEK293T were transfected with NF - kB reporter plasmid , A20 ( 3 ng ) or ABIN1 ( 15ng ) indicated concentrations of FLAG - tagged CARD14sh . Luciferase activity in lysates was measured 24 h later . Values are mean of triplicates ± s . e . expression of transfected plasmids was verified by western blot is shown in accompanying panels .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

(C) A20 and ABIN-1 inhibit CARD14-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid, A20 (3 ng) or ABIN1 (15ng) and the indicated concentrations of FLAG-tagged CARD14sh. Luciferase activity in cell lysates was measured 24 h later. Values are the mean of triplicates ± s.e. Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels.


( D ) A20 inhibits CARD14 - induced NF - kB activation through its ZnF domains . HEK293T were transfected with NF - kB reporter plasmid , FLAG - CARD14sh ( 20ng ) wild - type ( WT ) or mutant A20 expression plasmids ( 3ng ) as indicated . Luciferase activity in lysates was measured after 24 h . CARD14 A20 expression was measured by immunoblotting ( bottom panel ) . Data are representative of two independent experiments .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(D) A20 inhibits CARD14-induced NF-kB activation through its ZnF domains. HEK293T cells were transfected with NF-kB reporter plasmid, FLAG-CARD14sh (20ng) and wild-type (WT) or mutant A20 expression plasmids (3ng) as indicated. Luciferase activity in cell lysates was measured after 24 h. CARD14 and A20 expression was measured by immunoblotting (bottom panel). Data are representative of two independent experiments.


( A ) CARD14 ( E138A ) induces cytokine chemokine expression in human keratinocytes . were transfected with empty vector ( EV ) or CARD14 ( E138A ) as indicated . 24 h later , secretion of 36 different human cytokines , chemokines , acute phase was analyzed via a multiplex antibody array . was done using ImageJ software is expressed as relative CARD14 / EV values for a selected number of ( bottom panel ) .

O O O B-GENEPROD O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(A) CARD14(E138A) induces cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with empty vector (EV) or CARD14(E138A) as indicated. 24 h later, the secretion of 36 different human cytokines, chemokines, and acute phase proteins was analyzed via a multiplex antibody array. Quantification was done using ImageJ software and is expressed as relative CARD14/EV values for a selected number of proteins (bottom panel).


( B ) MALT1 deficiency inhibits CARD14 ( E138A ) - induced cytokine chemokine expression in human keratinocytes . were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 ( E138A ) . After 24 h , cytokine chemokine expression was analyzed is expressed as described in ( A ) .

O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(B) MALT1 deficiency inhibits CARD14(E138A)-induced cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14(E138A). After 24 h, cytokine and chemokine expression was analyzed and is expressed as described in (A).


( C ) Pathogenic CARD14 mutation enhances MALT1 - dependent CARD14 - induced IL - 8 expression in human keratinocytes . were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 wild - type ( WT ) , CARD14 ( G117S ) or CARD14 ( E138A ) as indicated . TNF treatment was used as a control . After 24 h , IL - 8 in supernatant was analyzed via ELISA . Values are mean of triplicates ± s . e . Expression of MALT1 CARD14 variants is verified by immunoblotting ( bottom panel ) .

O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O

(C) Pathogenic CARD14 mutation enhances MALT1-dependent CARD14-induced IL-8 expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14 wild-type (WT), CARD14(G117S) or CARD14(E138A) as indicated. TNF treatment was used as a control. After 24 h, IL-8 concentration in the cell supernatant was analyzed via ELISA. Values are the mean of triplicates ± s.e. Expression levels of MALT1 and CARD14 variants is verified by immunoblotting (bottom panel).


( D ) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14 - induced IL - 8 production A20 processing in human keratinocytes . were transfected with different CARD14 variants in presence or absence of mepazine as indicated . TNF treatment was used as a control . 24 h later , IL - 8 in supernatant were measured by ELISA . Values are mean of triplicates ± s . e . Expression of different CARD14 variants CARD14 - induced processing of endogenous A20 were analyzed by immunoblotting . Data are representative of two independent experiments . Significance , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t .

O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14-induced IL-8 production and A20 processing in human primary keratinocytes. Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated. TNF treatment was used as a control. 24 h later, IL-8 levels in the cell supernatant were measured by ELISA. Values are the mean of triplicates ± s.e. Expression of different CARD14 variants and CARD14-induced processing of endogenous A20 were analyzed by immunoblotting. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


A ) Treadmill ( n = 8 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by unpaired , two - Student ' s t * * * p = 0 . 005 ; * * * p = 0 . 0007 ( T3 ) * * * * p < 0 . 0001 ( T4 ) . Only p values for rapamycin - treated vs . untreated Cox15sm / sm mice are shown

O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O O B-ORGANISM O O

A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown


B ) Histological histochemical characterization of skeletal muscle in rapamycin - treated untreated Cox15sm / sm WT mice . H & E : hematoxylin eosin ; PAS : periodic acid Schiff reaction ; COX : cytochrome c oxidase ; SDH : succinate dehydrogenase . bars correspond to 50µm

O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O

B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm


C ) of cross - sectional of muscle in different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison : * * * p = 0 . 001 ; * * * p = 0 . 006 , * * * * p < 0 . 0001

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001


D ) of number of centralized nuclei in muscle ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * p = 0 . 0018 , * * p = 0 . 0052

O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O 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) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052


E ) of PAS - reaction in muscle ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * p = 0 . 0293 ( WT vs . Cox15sm / sm ) * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm Rapa )

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O

E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa)


F ) of COX - reaction in muscle ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * * * p < 0 . 0001 ; * * p = 0 . 0031

O O O B-SUBCELLULAR O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O 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) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031


G ) of SDH - reaction in muscle ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * p = 0 . 0199 , * * p = 0 . 0091

O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O 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) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091


A ) Spectrophotometric activities of respiratory chain ( n = 4 - 5 / group ) . CS : citrate synthase ; CIV : IV . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by two way ANOVA with Tukey ' s correction * * * * p < 0 . 0001 ( CS : WT vs . Cox15sm / sm ) , * * * p = 0 . 0010 ( CS : Cox15sm / sm vs . Cox15sm / sm rapamycin ) , * * * * p < 0 . 0001 ( CIV : WT vs . Cox15sm / sm ) , * * p = 0 . 0060 ( CIV / CS : WT vs . Cox15sm / sm )

O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O

A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm)


B ) Western blot immunovisualization of subunits of respiratory complexes . Note increased in Cox15sm / sm vs . WT samples , which are reduced to normal upon rapamycin treatment . Additional samples were run on a separate gel ( not shown )

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B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown)


C ) BNGE in gel activity for cIV . Sc : supercomplexes . Note that COX reaction is slightly increased in rapamycin - treated vs . untreated Cox15sm / sm samples

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O

C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples


D ) Immunoblot of 1D - BNGE using an anti - cIV antibody ( COX1 ) . Note that COX amount is slightly increased in rapamycin treated vs . untreated Cox15sm / sm . SDHB was used as a loading control

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O

D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control


E ) State III ( succinate - driven ) oxygen consumption ( n = 3 / group ) . Error bars represent . asterisks represent significance calculated by unpaired , two tail Student ' s t * * p = 0 . 0016 ( WT vs . Cox15sm / sm ) ; * p = 0 . 047 ( Cox15sm / sm vs . Cox15sm / sm rapamycin )

O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O

E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin)


F ) of mtDNA amount ( n = 9 / group ) . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey ' s correction : * * p = 0 . 0020 ; * p = 0 . 0296

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296


Note accumulation of profoundly altered mitochondria in Cox15sm / sm ( arrows ) , possibly as a result of partially digested organelles within endolysosomes . Black arrows indicate examples of normal mitochondria . In rapamycin - treated Cox15sm / sm only a few of these structures are present , which can be detected also in rapamycin - treated WT samples . bar corresponds to 1136 nm ( 4400X )

O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

Note the accumulation of profoundly altered mitochondria in Cox15sm/sm muscles (white arrows), possibly as a result of partially digested organelles within endolysosomes. Black arrows indicate examples of normal mitochondria. In rapamycin-treated Cox15sm/sm animals only a few of these structures are present, which can be detected also in rapamycin-treated WT samples. The bar corresponds to 1136 nm (4400X)


A ) Representative western blot of autophagy markers at steady state in skeletal muscle . GAPDH was used as a loading control . B ) Densitometric of immunoblots similar to that shown in A ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * p = 0 . 0068 ( LC3 - II WT vs . Cox15sm / sm ) , * * p = 0 . 0063 ( P62 WT vs . Cox15sm / sm ) * p = 0 . 025 ( P62 Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * p = 0 . 0098 ( S6 WT vs . Cox15sm / sm ) , * p = 0 . 034 ( P - S6 WT vs WT + rapamycin ) , * * * p = 0 . 0002 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * p = 0 . 014 ( P - S6 WT vs . WT + rapamycin ) * * * p = 0 . 0007 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin )

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O

A) Representative western blot of autophagy markers at steady state in skeletal muscle. GAPDH was used as a loading control. B) Densitometric analysis of immunoblots similar to that shown in A (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0068 (LC3-II WT vs. Cox15sm/sm), **p=0.0063 (P62 WT vs. Cox15sm/sm) *p=0.025 (P62 Cox15sm/sm vs. Cox15sm/sm+rapamycin), **p=0.0098 (S6 WT vs. Cox15sm/sm), *p=0.034 (P-S6 WT vs WT+rapamycin), ***p=0.0002 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), *p=0.014 (P-S6 WT vs. WT+rapamycin) ***p=0.0007 (Cox15sm/sm vs. Cox15sm/sm+rapamycin)


C ) of autophagic flux ( n = 3 / group ) . Upper panel : representative western blot of Cox15sm / sm WT treated with colchicine , rapamycin , or rapamycin plus colchicine . Note that LC3 - II was increased in Cox15sm / sm vs . WT samples . Colchicine did not increase LC3 - II suggesting a block in autophagic flux ; rapamycin plus colchicine treated Cox15sm / sm mice showed higher of LC3 - II , suggesting that rapamycin increased autophagic flux in Cox15sm / sm . Black : WT ; grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * p = 0 . 0049 ( WT vs . WT + colchicine ) , * * p = 0 . 0046 ( WT + rapamycin vs . WT + rapamycin + colchicine ) , * p = 0 . 0225 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0057 ( Cox15sm / sm colchicine vs . Cox15sm / sm + rapamycin + colchicine ) , * * * p = 0 . 0003 ( Cox15sm / sm rapamycin vs . Cox15sm / sm + rapamycin + colchicine )

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

C) Analysis of the autophagic flux (n=3/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rapamycin, or rapamycin plus colchicine. Note that LC3-II was increased in Cox15sm/sm vs. WT samples. Colchicine did not increase LC3-II levels suggesting a block in the autophagic flux; rapamycin plus colchicine treated Cox15sm/sm mice showed higher levels of LC3-II, suggesting that rapamycin increased the autophagic flux in Cox15sm/sm muscles. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0049 (WT vs. WT+colchicine), **p=0.0046 (WT+rapamycin vs. WT+rapamycin+colchicine), *p=0.0225 (WT vs. Cox15sm/sm), **p=0.0057 (Cox15sm/sm colchicine vs. Cox15sm/sm+rapamycin+colchicine), ***p=0.0003 (Cox15sm/sm rapamycin vs. Cox15sm/sm+rapamycin+colchicine)


A ) of autophagic flux ( n = 4 / group ) . Upper panel : representative western blot of Cox15sm / sm WT treated with colchicine , rilmenidine , or rilmenidine plus colchicine . Colchicine alone increased LC3 - II in WT but not Cox15sm / sm mice ; rilmenidine had no effect on WT LC3 - II , but reduced them in Cox15sm / sm mice . Rilmenidine plus colchicine increased LC3 - II in both WT Cox15sm / sm mice . Black : WT ; grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison : * * p = 0 . 0093 ( WT vs . WT + colchicine ) , * * * * p < 0 . 0001 ( WT + rilmenidine vs . WT + rilmenidine + colchicine ) , * * p = 0 . 0001 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0083 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine ) , * p = 0 . 0112 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine + colchicine ) , * * * * p < 0 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.000


B ) Treadmill ( n = 4 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent

O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O

B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM


C ) Histological histochemical characterization of skeletal muscle in rapamycin - treated untreated Cox15sm / sm WT mice . H & E : hematoxylin eosin ; GT : Gomori trichrome ; PAS : periodic acid Schiff reaction

C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction


D ) of cross - sectional of muscle in different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * * p = 0 . 0002 , * * * * p < 0 . 0001

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O 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) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001


E ) of number of centralized nuclei in muscle fibres ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * p = 0 . 0091

O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O 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 number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091


A ) anti - TFEB immunofluorescence on rapamycin - rilmenidine - treated WT Cox15sm / sm vs . untreated ( UT ) samples . CD11C ( blue signal ) indicates inflammatory . Right panel : of n = 3 / group . scale bars correspond to 30µm . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison B ) of TFEB - positive nuclei ( n = 3 / group ) . was performed using Imaris spots surface excluding CD11C positive nuclei . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * p = 0 . 0199 ( WT vs . WT + rapamycin ) , * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * * p = 0 . 0001

O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O

A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001


A ) Real - time PCR of Lamp1 transcript ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent . asterisks represent significance calculated by Student ' s t * * p = 0 . 008 , * p = 0 . 038

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O 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) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038


B ) Anti - LAMP1 in rapamycin - rilmenidine - treated WT Cox15sm / sm vs . untreated ( UT ) samples . Rapamycin , but not rilmendine , increases number of LAMP1 - positive vesicles in Cox15sm / sm samples ( inset ) . scale bars correspond to 30µm

O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O

B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm


C ) of LAMP1 - positive vesicles ( n = 3 / group ) . Error bars represent . asterisks represent significance calculated by one - way ANOVA with Tukey post - hoc multiple comparison * * * * p < 0 . 0001

O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O 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 LAMP1-positive vesicles (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001


D ) Cathepsin L specific activity ( n = 3 / group ) . Error bars represent . asterisks represent significance calculated by Student ' s t * p = 0 . 0133

O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O 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) Cathepsin L specific activity (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test *p=0.0133


( C ) Cleavage of supercoiled ( SC ) plasmid substrates using purified Cas12l nuclease respective sgRNA variants . Efficient full linearization ( FLL ) of substrate resulting from a complete double - strand break was only observed when using sgRNAs bearing 5 ' most end of tracrRNA ( variants 1 3 ) . Moreover , terminator - like Hairpin 3 is not required for target cleavage ( variant 1 ) . OC - open circular ; FLL - full linear ; SC - supercoiled .

O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.


( B ) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within RuvC nuclease domain of Asp2Cas12l Asp3Cas12l . wt - wildtype .

O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O

(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.


( C ) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition . molar of RNP to substrate was kept low ( 5 : 1 ) to increase reaction stringency . Asp2Cas12l predominantly recognizes a 5 ' - CCY - 3 ' PAM Asp3Cas12l a 5 ' - CCB - 3 ' PAM . N = 3 . Data information : In ( C ) , data points are plotted , where n = 4 replicates from independent experiments ( Asp2Cas12l ) n = 3 replicates from independent experiments ( Asp3Cas12l ) . data points were fitted to a single exponential association curve ( solid lines ) .

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).


( A ) Asp2 Asp3Cas12l RNP complexes degrade M13 ssDNA in presence of single - stranded ( ss ) or dsDNA ( including PAM ) activators with target sequences complementary to gRNA spacer . NS activator - non specific activator ( ssDNA oligonucleotide or dsDNA duplex ) with no complementarity to spacer of gRNA .

O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.


( B ) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched ssDNA or ssRNA probes . Background - subtracted traces from reporter assays with favored ssDNA ( 5 ' - CCCCCCCC - 3 ' ) or ssRNA ( 5 ' - CCCCCCCC - 3 ' ) probes . RFU - relative units . ( C ) Background - subtracted traces of reporter cleavage using ssDNA or ssRNA probes . ( D ) Rates of trans - degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets . Collateral ssDNAse activity is about 3 - fold higher than rate of ssRNA degradation . intensities in B - C were normalized against of a reaction containing only probe to account for imperfect quenching or degradation of reporters . Data information : In ( B - D ) , data points from n = 3 replicates from independent experiments are plotted . In ( D ) , bars represent mean .

O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O 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-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes. (D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.


Distribution of 25 - 35 mer reads mapping to mouse genome . Proportion of 30 - 35 nt genomic reads matching mouse tRNA sequences . sequencing was done in duplicates . Error bars represent , significance was calculated using unpaired t - .

O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.


Perbase coverage of 30 - 35 nt reads across parental tRNA . Anticodon positions are demarcated by discontinuous lines ( Blue = LIF - treated , Grey = Wnt3 + LIF - treated , Red = RA - treated mESCs ) . Perbase coverage of 30 - 35 nt reads across 468 parental tRNAs plotted as heatmaps .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.


Northern blot of candidate tRNAs tsRNAs tsGlnCTG , tsGlyGCC , tsGluTTC , tsValCAC tsLysTTT . black arrows represent tRNAs red arrows mark tsRNAs .

Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.


Northern Blot of 5 ' - tsRNAs ; tsGlyGCC , tsGlnCTG , tsGluTTC , tsLysTTT , tsValCAC at various timepoints of RA - induced mESCs differentiation showing dynamic expression of these 5 ' - tsRNAs . Relative of tsRNA bands from northern hybridization tested across two biological replicates . solid lines represent expression of tsRNAs dashed lines represent tRNAs .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O

Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.