D N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline overnight , and cells were treated with TSI for indicated time , followed by UBA - pull down . Representative of three independent experiments . E N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 16 hrs . Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline . Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 ( BAF ) , PS341 ( PS ) or left untreated ( UT ) . IDN - 6556 was added to all conditions to block apoptosis . Cells were collected 0 , 2 , 4 , 6 hrs after medium replacement , followed by UBA - pull down . Representative of three independent experiments .

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

D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.


B WT and 4KR mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 6 hrs and cells were untreated ( UT ) or treated with TSI , followed by UBA - pull down . Representative of three independent experiments .

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B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.


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 .

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

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 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 ) 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 . ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 ) .

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(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 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 .

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(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 ) 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 . ) .

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

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


( b ) Immunofluorescence of HeLa cells 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 cells coexpressing HA - Nod1 ( red ; c ) or HA - Nod2 ( red ; d ) and 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 cells expressing a Flag - tagged truncated form of ATG16L1 ( Flag - ΔN85 - ATG16L1 ; green ) . Blue , DAPI .

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


( f ) Immunofluorescence of HeLa cells coexpressing HA - Nod2 ( red ) and 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 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 .

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(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 ) 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 ) .

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(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 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 .

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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 ) 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 . ) .

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(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 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 .

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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 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 .

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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 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 .

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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 cells 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 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 . ) .

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(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 ) 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 .

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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 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 .

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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 distance travelled ( G ) and 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 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 .

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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 the heart and 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 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 .

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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 and diaphragm muscle architecture was analyzed by H & E staining . 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 the diaphragm via Masson Trichrome staining . Scale bars , 100 μm .

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


WT , Dup18 - 30 untreated and Dup18 - 30 treated mice were tested 7 weeks 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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(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 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 .

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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 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 ) .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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

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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 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

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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 ) 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

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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 ) 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

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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 ) 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 )

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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 ) 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

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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 ) 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

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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 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 )

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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 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 )

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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 the COX reaction is slightly increased in rapamycin - treated vs . untreated Cox15sm / sm samples

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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 muscles . SDHB was used as a loading control

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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 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 )

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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 ) 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

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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 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 )

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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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 ) 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 )

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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 ) 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 .

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

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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 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

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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 ) 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

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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 ) 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

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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 - 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

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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 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

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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 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

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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 ) 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

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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 SEM . The asterisks represent the significance levels calculated by Student ' s t test * p = 0 . 0133

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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 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 .

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(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 the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l . wt - wildtype .

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(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 . 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 ) .

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(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 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 .

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(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.