Figure 4 ( A ) PAS staining for total glycogen in skin fibroblasts from different APBD patients . Staining fluorescence was quantified by InCell2200 ( see Methods ) . Means ( + / - SEM ) are statistically different from each other ( p < 0 . 0001 , One Way ANOVA ) . n = 3 technical replicates from each patient indicated at the x - axis were used . ( B ) PAS staining for total glycogen in APBD87 fibroblasts glucose - starved for 48 h ( left ) , or glucose starved and then replenished for the last 24 h to induce glycogen burden ( right ) . Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter . Scale bar , 100 µm . ( D ) Glycolytic ( red ) and mitochondrial ( blue ) ATP production determined by Agilent ' s Seahorse machine and real - time ATP rate assay kit . HC and APBD patient fibroblasts were serum / glucose - starved for 48 h and then full medium was replenished for 24 h without ( untreated ) , or with ( chronic ) 50 µM 144DG11 . Acute , 50 µM 144DG11 was added on assay for 20 min after 24 h of serum / glucose replenishment . Readings were normalized to cell number as determined by Crystal Violet staining . Shown are mean and SEM values based on n = 3 - 6 technical repeats . For all experiments ( HC , p < 0 . 01 ; APBD1 , p < 0 . 02 ; APBD2 , p < 0 . 09 ; APBD3 , p < 0 . 11 ; One Way ANOVA with Sidak ' s post - hoc correction for multiple comparisons ) glycolytic ATP production was increased , as compared to untreated control , by acute , but not chronic , supplementation of 144DG11 . Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts ( p < 0 . 06 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) PAS staining for total glycogen in skin fibroblasts from different APBD patients. Staining fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient indicated at the x-axis were used.(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm.(D) Glycolytic (red) and mitochondrial (blue) ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11 was added on assay for 20 min after 24 h of serum/glucose replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).


Figure 5 ( C ) Cellular thermal shift assay ( CETSA ) of different targets of the 144DG11hetero - assembly ( B ) . Only LAMP1 was significantly protected by 144DG11 from heat - mediated denaturation ( see right shift in its Tm in the lower panel ) suggesting its specific interaction with 144DG11 . n = 3 biological replicates . Error bars represent S . D .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.


Figure 6 ( A ) Left panel , Autophagic flux , determined by the extent of lysosomal inhibitors - dependent increase in the ratio of lipidated to non - lipidated LC3 ( LC3II / LC3I ) , is increased by 144DG11 ( 50µM , 24h ) . Right panel , 144DG11 - mediated increase in autophagic flux , demonstrated by enhanced degradation of the autophagy substrate p62 . Shown are representative immunoblots ( grouped from different gels ) and densitometric quantifications + / - s . d . ( for LC3II / LC3I , n = 4 biological replicates , * p < 0 . 0023 ; for p62 , n = 5 biological replicates , * p < 0 . 0099 ; two - tailed t - test ) . ( B ) Left panel , TEM images of liver tissue from vehicle or 144DG11 - treated Gbeys / ys mice . High - magnification ( right , scale bars = 200 nm ) and low - magnification ( left , scale bars = 1 µm ) images show higher levels of glycogen / polyglucosan in lysosomes and cytosol , respectively . Right panel , quantification ( + / - s . d ) of lysosomal glycogen particles ( n = 3 biological replicates , * p < 0 . 03 , two - tailed t - test ) . G , Glycogen / polyglucosan ; L , Lysosomes ; M , Mitochondria . ( C ) 144DG11 reduces LC3 and p62 in mouse liver , but not muscle ( n = 3 biological replicates , * p < 0 . 04 for LC3 , * * p < 0 . 0007 for p62 ; two - tailed t - test , SEM ) . Scale bars , 10 µm ( E ) LAMP1 - KD and 144DG11 treatment cause lysosomal acidification . Upper panel , flow cytometry results showing that 144DG11 slightly increased acidification ( yellow to blue median fluorescence ratio ( Y / B ) ) in control , GFP - transduced , APBD fibroblasts ( Y / B ( GFP / 144DG11 ) > Y / B ( GFP ) , p < 0 . 12 ) , but significantly acidified LAMP1 - KD , GFP - shLAMP1 - transduced , APBD fibroblasts ( Y / B ( LAMP1 - KD / 144DG11 ) > ( Y / B ( LAMP1 - KD ) , p < 0 . 03 ) . LAMP1 - KD itself led to the most significant acidification ( Y / B ( LAMP1 - KD ) > Y / B ( GFP ) , p < 0 . 007 ) . n = 3 , two - tailed t - tests . Middle panel Lysosensor staining of the corresponding cells . Yellow fluorescence intensity correlates with acidification . Lower panel , PAS ( glycogen ) staining of the corresponding cells . Scale bars , 50 µm . ( G ) 144DG11 reduces lysosomal ( LAMP1 positive ) area in liver , but not muscle , of Gbeys / ys mice ( n = 3 biological replicates , * p < 0 . 01 , two - tailed t - test , SEM ) . Scale bars , 5 µm for liver ( upper left panel ) , and 10 µm for muscle ( lower left panel ) . Right panel shows quantification of the left panel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).(B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.(C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm(E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm.(G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.


Figure 7 ( B ) Morphological characterization of lysosomes based on lysotracker staining of n = 4 HC and n = 4 ( biological replicates ) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h . Values are deviations from untreated HC . 144DG11 has reduced lysotracker mean intensity , integrated intensity ( DXA ) , and area in both HC and APBD fibroblasts ( * * * p < 0 . 0001 , One - Way ANOVA with Sidak ' s multi - comparison post - hoc test ) . ( C ) Functional ( mitochondrial membrane potential ( TMRE parameters ) ) and biomass ( mitotracker ) characterization of mitochondria in HC and APBD cells treated and analyzed as in ( B ) . Mean ( * * * , p < 0 . 0001 ) and integrated ( * * * , p < 0 . 0006 ) TMRE intensities , and mitochondrial biomass ( * * * , p < 0 . 0001 ) , were reduced in APBD v HC . 144DG11 increased TMRE mean ( * * , p < 0 . 009 , * * , p < 0 . 006 ) and integrated ( * , p < 0 . 01 , * * , p < 0 . 001 ) intensities in APBD and HC cells , respectively and mitotracker intensity in APBD cells only ( * * * , p < 0 . 0001 ) . Data represent n = 3 biological replicates . Statistical differences analyzed by One - way ANOVA with multiple comparisons .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Morphological characterization of lysosomes based on lysotracker staining of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test).(C) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE intensities, and mitochondrial biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD and HC cells, respectively and mitotracker intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons.


Figure 1A Agarose gel image illustrating PCR amplicons of ltaS ( LBA0447 ) , slpB , and slpX deletions in NCK2187 . B SDS - PAGE gel of 5 M LiCl ‐ purified S ‐ layer fractions from the parental strains , NCK56 and NCK1909 , NCK2030 ( LTA + , SlpA + , SlpB − , SlpX − ) , and NCK2187 ( LTA − , SlpA + , SlpB − , SlpX − ) . C B6 mice were orally gavaged with 109 CFU erythromycin ‐ resistant NCK56 or NCK2187 . Fecal pellets were collected daily and tested for the presence of erythromycin ‐ resistant strains . n = 3 mice / group . Data are representative of five independent experiments and are shown as mean ± SEM . D ColonicLPL cells were co ‐ cultured with NCK56 or NCK2187 ( 1 : 1 ) , and secreted cytokines were measured in the supernatants . Data are shown as mean ± SEM and are representative of two experiments performed in triplicate . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.D ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.


Figure 2A B6 mice were orally gavaged with 109CFU NCK56 ( blue ) or NCK2187 ( green ) on days 0 , 3 , 6 , and 9 , or left untreated , and immune responses in the colon analyzed at day 14 by flow cytometry . B , C B6 FoxP3 ‐ GFP mice were treated and evaluated as in ( A ) . Regulatory cytokine production in FoxP3 ‐ GFP + ( green ‐ dotted bars ) versus FoxP3 ‐ GFP − ( white bars ) cells was measured by intracellular staining and FACS analyses ( C ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).


Figure 3A B6 Rag1 − / − mice were injected with 106CD4 + CD45RBhiT cells and then orally gavaged with NCK56 ( red ) , NCK2187 ( green ) , or SlpA ( blue ) , 1 and 3 days after transfer , and subsequently once a week for four consecutive weeks , or left untreated ( magenta ) . A group of mice was co ‐ transferred with CD4 + CD25 + T cells as a positive control for protection ( Tregs ; gray ) . Colitis severity was determined in part by weight loss , diarrhea scores , and FOB . See Supplementary Tables S1 , S2 and S3 for statistical analyses results . B , C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in ( A ) were also used as measures of disease . Scale bar = 50 μm . D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in ( A ) , or sham adoptive transferred ( white bars ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A B6 Rag1−/−mice were injected with 106CD4+CD45RBhiT cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).


Figure 4Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Colonic expression of tight junction ‐ associated genes Cldn3 and Ocln , determined by RT - PCR relative to 18S rRNA ( A ) , were used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice . Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Passive transepithelial absorption of FITC ‐ dextran ( B ) , was used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( white bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three individual experiments and are shown as mean ± SEM . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , and red asterisks to NCK56 ‐ treated mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


Figure 5A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187 , and the colonic gene expression of C ‐ type lectin receptors were measured by RT - PCR . Each box represents an individual mouse ; n = 4 . Data represent three individual experiments . B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; blue = SlpA ‐ coated beads + SIGNR1 ‐ hFc ; red = SlpA ‐ coated beads + SIGNR3 ‐ hFc . Binding assay results were confirmed five independent times . C Binding of SlpA to SIGNR3 , but not SIGNR1 expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; green = SIGNR1 ‐ transfected CHO ‐ S cells + labeled SlpA ; red = SIGNR3 ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times . D Binding of SlpA to recombinant human DC ‐ SIGN / CD209 ‐ Fc chimera was analyzed by flow cytometry . Gray tinted line = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion protein ; red = SlpA ‐ coated beads + DC ‐ SIGN ‐ Fc . Binding assays were performed and confirmed three times . E Binding of SlpA to DC ‐ SIGN expressed by CHO ‐ S cells . Gray tinted line = untransfected CHO ‐ S cells ; blue = untransfected CHO ‐ S cells + labeled SlpA ; red = DC ‐ SIGN ‐ transfected CHO ‐ S cells + labeled SlpA . Binding assays in CHO ‐ S cells were performed three individual times . F , G IL ‐ 1β production by colonic DCs of naïve WT B6 GF or B6 Signr3 − / − ( KO ) mice treated with NCK56 ( blue ) or NCK2187 ( green ) four times , or left untreated ( black ) , as determined by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM . * P 0 . 05 . Black asterisks compare NCK2187 to untreated ( PBS ) mice , and red asterisks to NCK56 ‐ treated mice . H Frequency of colonic FoxP3 + Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry . n = 5 mice / group . Data represent four individual experiments and are shown as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT-PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.F, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.


Figure 6WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . A Colitis severity was determined in part by weight loss . n = 5 mice / group . WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . B , C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease . Scale bar = 50 μm . n = 5 mice / group . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA . WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . D Colonoscopies were performed in the different groups with a Multi ‐ Purpose Rigid ™ Telescope attached to a TELE PACK X on day 10 . WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . E Mean relative colonic expression of tight junction ‐ associated genes in WT mice . n = 5 mice / group . WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 and − 1 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression . F Fecalalbumin levels in WT mice as a measure of intestinal permeability . n = 5 mice / group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.F Fecalalbumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.


Figure 7Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS ‐ treated WT ( left ) and KO mice ( right ) . Empty bars = WT ; lined bars = KO ; white bars = untreated ; purple bars = DSS ; red bars = DSS + NCK56 ; green bars = DSS + NCK2187 ; blue bars = DSS + SlpA . Signr3 + / + ( WT ) or Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 1 and − 3 , and 3 % DSS was given in the drinking water . Mice were gavaged with bacteria or purified SlpA every other day for an additional three times , and immunity was analyzed by flow cytometry at day 10 . B , C Colonic DCs and macrophages ( MΦs ) were analyzed by flow cytometry for the production of IL ‐ 1β ( B ) , and colonic FoxP3 + Tregs were evaluated for co ‐ expression of RORγt + ( C ) . n = 5 mice / group . Gray tinted line = isotype control ; black = untreated ; purple = DSS ; red = DSS + NCK56 ; green = DSS + NCK2187 ; blue = DSS + SlpA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.


Figure 1 . The violin plot of ACE2 gene expression of all major cell types .

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Figure 1.The violin plot of ACE2 gene expression of all major cell types.


Figure 2 . Cell type annotations of all four patients by the tSNE plot . Hepatocyte - cholangiocytes cluster includes potential stem - like cells capable of differentiating into both cell types . B ) tSNE plot colored by patientsHeatmap of marker genes of 16 cell types identified . The red box highlights the cell type specific expression of ACE2 . The violin plot of ACE2 gene expression across all cell types .

O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O

Figure 2.Cell type annotations of all four patients by the tSNE plot. Hepatocyte-cholangiocytes cluster includes potential stem-like cells capable of differentiating into both cell types. B) tSNE plot colored by patientsHeatmap of marker genes of 16 cell types identified. The red box highlights the cell type specific expression of ACE2.The violin plot of ACE2 gene expression across all cell types.


Figure 1A , B NAD and nicotinamide ( NAM ) levels in heart of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 5 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . C , D NAD and NAM levels in diaphragm of WT ( n = 5 , n = 6 , respectively ) , mdx ( n = 4 , n = 5 , respectively ) and mdx / CD38 - / - mice ( n = 6 , n = 5 , respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; B : Kruskal - Wallis followed by Dunn ' s test ; D : Kruskal - Wallis followed by Mann - Whitney tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E , F levels of ADP - ribose ( ADPR ) ( E ) , cyclic ADP - ribose ( cADPR ) ( F ) expressed as nmol / mg protein in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) qPCR analysis of mRNA levels of Sterile alpha and Toll / interleukin - 1 receptor motif - containing 1 ( SARM1 ) in heart of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - mice ( n = 5 , n = 5 , n = 6 respectively ) . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . H , I Levels of ADPR ( H ) , cADPR ( I ) expressed as nmol / mg protein in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; I : Welch ' s ANOVA followed by Welch ' s t - tests Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( J ) qPCR analysis mRNA levels of SARM1 in diaphragm of WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : J : ANOVA Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . K , L Western blot analysis of CD38 protein expression in heart ( K ) and diaphragm ( L ) of WT ( n = 5 , n = 6 , respectively ) and mdx ( n = 5 ) mice . Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38 / vinculin . Data information : Each dot of the graphs represents a value / mouse . After normality and variance comparison tests , significance was assessed using : unpaired Student ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . M , N qPCR analysis of CD38 mRNA levels in heart ( M ) and diaphragm ( N ) of WT ( n = 6 ) and mdx ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests , significance was assessed using : M : unpaired Student ' s t - test ; N : unpaired Welch ' s t - test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A,B NAD and nicotinamide (NAM) levels in heart of WT (n=5, n=6, respectively), mdx (n=5, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). C,D NAD and NAM levels in diaphragm of WT (n=5, n=6, respectively), mdx (n=4, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; B: Kruskal-Wallis followed by Dunn's test; D: Kruskal-Wallis followed by Mann-Whitney tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E,F levels of ADP-ribose (ADPR) (E), cyclic ADP-ribose (cADPR) (F) expressed as nmol/mg protein in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.(G) qPCR analysis of mRNA levels of Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) in heart of WT (n=6), mdx (n=5) and mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.H,I Levels of ADPR (H), cADPR (I) expressed as nmol/mg protein in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; I: Welch's ANOVA followed by Welch's t-tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.(J) qPCR analysis mRNA levels of SARM1 in diaphragm of WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: J: ANOVA Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.K, L Western blot analysis of CD38 protein expression in heart (K) and diaphragm (L) of WT (n=5, n=6, respectively) and mdx (n=5) mice. Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38/vinculin. Data information: Each dot of the graphs represents a value/mouse. After normality and variance comparison tests, significance was assessed using: unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.M, N qPCR analysis of CD38 mRNA levels in heart (M) and diaphragm (N) of WT (n=6) and mdx (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: M: unpaired Student's t-test; N: unpaired Welch's t-test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 2A Cardiac function evaluated by echocardiography in mdx / CD38 - / - mice : the dot plots show the left ventricular ( LV ) diastolic ( A1 ) and systolic ( A2 ) inner diameters and LV ejection ( A3 ) fraction in 7 - month - old WT ( n = 5 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 10 ) mice . Data information : : Each dot of the graphs represents a mouse . in duplicate After normality and variance comparison tests , significance was assessed using : A1 ANOVA followed by Fisher ' s LSD test ; A2 , A3 Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( B1 ) and cardiac troponin I ( cTnI ) ( B2 ) in 7 - month - old WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 11 ) mice . Data information : : Each dot of the graphs represents a mouse . After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Cropped images revealed the collagen ( blue ) stained by Masson ' s trichrome staining in the heart of WT , mdx , and mdx / CD38 - / - mice . Dot plot showing the quantification of heart collagen staining area ( % total area ) in 7 - month - old WT , mdx , and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : : Each dot of the graphs represents a mouse . in triplicate After normality and variance comparison tests , significance was assessed using ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D Isoproterenol - induced heart failure in 3 - month - old mice . The Kaplan - Meier curve shows the survival rate of mdx mice ( NaCl , n = 5 ) ; mdx ( n = 19 ) and mdx / CD38 - / - ( n = 9 ) mice following isoproterenol ( subcutaneous injection ) at 2 . 5 mg / kg / d for 10 days . Data information : one value / mouse . After normality and variance comparison tests , significance was assessed using D : Log - rank Mantel - Cox test and Log - rank test for trend and Gehan - Breslow - Wilcoxon test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E Histogram showing isoproterenol - induced heart hypertrophy in mdx NaCl ( n = 5 ) mice , surviving mdx ( n = 9 ) , mdx / CD38 - / - NaCl ( n = 9 ) and mdx / CD38 - / - ( n = 9 ) mice , expressed as heart weight / body weight ratio ( HW / BW ) . Data information : one value / mouse . After normality and variance comparison tests , significance was assessed using Kruskal - Wallis followed by Mann - Whitney tests Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . F Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( F1 ) and cardiac troponin I ( cTnI ) ( F2 ) following isoproterenol treatment in mdx NaCl ( n = 5 ) , surviving mdx ( n = 8 ) , mdx / CD38 - / - NaCl ( n = 5 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : in duplicate After normality and variance comparison tests , significance was assessed using F2 Kruskal - Wallis followed by Mann - Whitney tests ; F1 : ANOVA followed by Student ' s / Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . G , H Time - lapse images recorded by confocal microscopy in " line scanning mode " , showing Ca2 + sparks and waves in cardiomyocytes at rest , extracted from mdx and mdx / CD38 - / - mice ; scale bars : 10 µM ( horizontal ) , 500 ms ( vertical ) . Bar graphs showing the averaged Ca2 + sparks ( G ) and waves ( H ) frequencies in cardiomyocytes isolated from WT ( n = 21 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 33 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . J Bar graph showing the post - rest potentiation in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Cardiac function evaluated by echocardiography in mdx/CD38-/- mice: the dot plots show the left ventricular (LV) diastolic (A1) and systolic (A2) inner diameters and LV ejection (A3) fraction in 7-month-old WT (n=5), mdx (n=6) and mdx/CD38-/- (n=10) mice. Data information: : Each dot of the graphs represents a mouse. in duplicate After normality and variance comparison tests, significance was assessed using:A1 ANOVA followed by Fisher's LSD test; A2,A3 Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (B1) and cardiac troponin I (cTnI) (B2) in 7-month-old WT (n=7), mdx (n=6) and mdx/CD38-/- (n=11) mice. Data information: : Each dot of the graphs represents a mouse. After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.C Cropped images revealed the collagen (blue) stained by Masson's trichrome staining in the heart of WT, mdx, and mdx/CD38-/- mice. Dot plot showing the quantification of heart collagen staining area (% total area) in 7-month-old WT, mdx, and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: : Each dot of the graphs represents a mouse. in triplicate After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.D Isoproterenol-induced heart failure in 3-month-old mice. The Kaplan-Meier curve shows the survival rate of mdx mice (NaCl, n=5); mdx (n=19) and mdx/CD38-/- (n=9) mice following isoproterenol (subcutaneous injection) at 2.5 mg/kg/d for 10 days. Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using D: Log-rank Mantel-Cox test and Log-rank test for trend and Gehan-Breslow-Wilcoxon test ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E Histogram showing isoproterenol-induced heart hypertrophy in mdx NaCl (n=5) mice, surviving mdx (n=9), mdx/CD38-/- NaCl (n=9) and mdx/CD38-/- (n=9) mice, expressed as heart weight/body weight ratio (HW/BW). Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Mann-Whitney tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.F Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (F1) and cardiac troponin I (cTnI) (F2) following isoproterenol treatment in mdx NaCl (n=5), surviving mdx (n=8), mdx/CD38-/- NaCl (n=5) and mdx/CD38-/- (n=8) mice. Data information: in duplicate After normality and variance comparison tests, significance was assessed using F2 Kruskal-Wallis followed by Mann-Whitney tests; F1: ANOVA followed by Student's/ Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.G,H Time-lapse images recorded by confocal microscopy in "line scanning mode", showing Ca2+ sparks and waves in cardiomyocytes at rest, extracted from mdx and mdx/CD38-/- mice; scale bars: 10 µM (horizontal), 500 ms (vertical). Bar graphs showing the averaged Ca2+ sparks (G) and waves (H) frequencies in cardiomyocytes isolated from WT (n=21 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT (n=24 cells), mdx (n=33 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.J Bar graph showing the post-rest potentiation in cardiomyocytes from WT (n=24 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 3A Measurement of the ventilatory mechanic by barometric plethysmography : dot plots showing inspiratory ( A1 ) and expiratory times ( A2 ) , the relaxation time ( A3 ) and the respiratory frequency ( A4 ) in WT ( n = 8 ) , mdx ( n = 11 ) and mdx / CD38 - / - ( n = 9 ) mice . Data information : Each dot of the graphs represents a mouse . one value / mouse After normality and variance comparison tests , significance was assessed using : A1 , A3 ANOVA followed by Fisher ' s LSD test ; A2 , A4 : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B Images showing muscle fiber typology revealed by immunostaining . Localization of the slow MyHC ( Type I ) fiber and fast MyHCs ( Type IIa and IIb / X ) fibers , along with laminin ( red ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 100 µm . Histogram showing the percentage of I , IIa and IIb / X fiber type distribution in diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : ; B : Chi - square test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Fiber size distribution in the diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : C : Kolmogorov - Smirnov test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D Images revealing the collagen ( blue ) by Masson ' s trichrome staining in diaphragm of WT , mdx , and mdx / CD38 - / - mice . Quantification of collagen staining area ( % total area ) in the diaphragm of WT , mdx and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : Each dot of the graphs represents a mouse . D in duplicate ; After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E Embryonic myosin expression : immunostaining showing its localization along with laminin ( green ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 50 µm . Histogram showing the relative proportion of embryonic myosin area ( % total area ) in diaphragm of WT ( n = 7 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : Each dot of the graphs represents a mouse . E one value / mouse . After normality and variance comparison tests , significance was assessed using : E : Welch ' s ANOVA followed by Welch ' s t - tests . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Measurement of the ventilatory mechanic by barometric plethysmography: dot plots showing inspiratory (A1) and expiratory times (A2), the relaxation time (A3) and the respiratory frequency (A4) in WT (n=8), mdx (n=11) and mdx/CD38-/- (n=9) mice. Data information: Each dot of the graphs represents a mouse. one value/mouse After normality and variance comparison tests, significance was assessed using: A1,A3 ANOVA followed by Fisher's LSD test; A2, A4 : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B Images showing muscle fiber typology revealed by immunostaining. Localization of the slow MyHC (Type I) fiber and fast MyHCs (Type IIa and IIb/X) fibers, along with laminin (red) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 100 µm. Histogram showing the percentage of I, IIa and IIb/X fiber type distribution in diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: ; B: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.C Fiber size distribution in the diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: C: Kolmogorov-Smirnov test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.D Images revealing the collagen (blue) by Masson's trichrome staining in diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E Embryonic myosin expression: immunostaining showing its localization along with laminin (green) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 50 µm. Histogram showing the relative proportion of embryonic myosin area (% total area) in diaphragm of WT (n=7), mdx (n=8) and mdx/CD38-/- (n=8) mice. Data information: Each dot of the graphs represents a mouse. E one value/mouse. After normality and variance comparison tests, significance was assessed using: E: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 4A Images showing immunostaining of myeloid cells showing F4 / 80 ( macrophage marker ) , Ly - 6G / 6C ( monocyte , granulocyte and neutrophil marker ) , CD8 ( cytotoxic T lymphocyte marker ) and IL - 6 positive cells in diaphragm of 7 - month - old WT ( n = 8 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 7 or 8 ) mice . Lower panel : histograms quantifying the percentage of F4 / 80 ( A1 ) mdx / CD38 - / - ( n = 7 ) , LY6 ( A2 ) mdx / CD38 - / - ( n = 8 ) , CD8 ( A3 ) mdx / CD38 - / - ( n = 7 ) , IL - 6 ( A4 ) mdx / CD38 - / - ( n = 8 ) positive cells . Scale bars : 50 µm . Data information : Each dot of the graphs represents a mouse . A one value / mouse After normality and variance comparison tests , significance was assessed using : A1 - 4 : Welch ' s ANOVA followed by Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B qPCR analysis of mRNA levels of interleukin - 1β ( IL - 1ß ) ( B1 ) and - 6 ( IL - 6 ) ( B2 ) , cyclin - dependent kinase inhibitor 1 ( p21 ) ( B3 ) transforming growth factor - β ( TGF - β ) ( B4 ) , and senescence markers ( cell - cycle inhibitor p16 , INK4a ) ( B5 ) , Collagen Type I Alpha 1 Chain ( Col1a1 ) ( B6 ) in diaphragm of 20 - month - old WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse . B in duplicate . After normality and variance comparison tests , significance was assessed using : B1 - 6 : ANOVA followed by Fisher ' s LSD test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Images showing immunostaining of myeloid cells showing F4/80 (macrophage marker), Ly-6G/6C (monocyte, granulocyte and neutrophil marker), CD8 (cytotoxic T lymphocyte marker) and IL-6 positive cells in diaphragm of 7-month-old WT (n=8), mdx (n=8) and mdx/CD38-/- (n=7 or 8 ) mice. Lower panel: histograms quantifying the percentage of F4/80 (A1) mdx/CD38-/- (n=7), LY6 (A2) mdx/CD38-/- (n=8), CD8 (A3) mdx/CD38-/- (n=7), IL-6 (A4) mdx/CD38-/- (n=8) positive cells. Scale bars: 50 µm. Data information: Each dot of the graphs represents a mouse. A one value/mouse After normality and variance comparison tests, significance was assessed using: A1-4: Welch's ANOVA followed by Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B qPCR analysis of mRNA levels of interleukin-1β (IL-1ß) (B1) and -6 (IL-6) (B2), cyclin-dependent kinase inhibitor 1 (p21) (B3) transforming growth factor-β (TGF-β) (B4), and senescence markers (cell-cycle inhibitor p16, INK4a) (B5), Collagen Type I Alpha 1 Chain (Col1a1) (B6) in diaphragm of 20-month-old WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse. B in duplicate. After normality and variance comparison tests, significance was assessed using: B1-6: ANOVA followed by Fisher's LSD test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 5A NAD + levels in limb muscles of 20 - month - old WT ( n = 7 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 6 ) mice . Data information : Each dot of the graphs represents a mouse . in duplicate ; After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B CD38 expression : western blot analysis of CD38 ( B1 ) protein in limb of WT and mdx mice ( n = 6 per group ) . Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38 / vinculin . qPCR analysis of CD38 mRNA ( B2 ) in limb of 20 - month - old WT and mdx mice ( n = 4 per group ) . Data information : Each dot of the graphs represents a mouse . one value / mouse ; After normality and variance comparison tests , significance was assessed using : B1 : unpaired Welch ' s t - test ; B2 : unpaired Student ' s t - test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C Histograms showing the grip duration ( latency to fall ) ( C1 ) and the limb force ( C2 ) measured by a grip test in WT ( n = 89 ) , mdx ( n = 52 , 53 respectively ) and mdx / CD38 - / - ( n = 58 ) mice ( age : 9 to 26 months old ) . Data information : C1 one value / mouse ; C2 in triplicate . After normality and variance comparison tests , significance was assessed using : : unpaired Student ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . D Images showing muscle fiber typology revealed by immunostaining . Localization of the slow MyHC ( Type I ) fiber and fast MyHCs ( Type IIa and IIb / X ) fibers , along with laminin ( red ) on transverse cross - sections from soleus and tibialis ( TA ) of WT , mdx and mdx / CD38 - / - mice . Scale bars : 100 µm . Histogram showing the percentage of I , IIa and IIb / X fiber type distribution in soleus and TA of WT , mdx and mdx / CD38 - / - mice . Experiments performed in WT ( n = 3 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 7 ) mice for soleus ; WT ( n = 5 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 6 ) mice for TA . Data information : one value / mouse ; After normality and variance comparison tests , significance was assessed using : D : Chi - square test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E Fiber size distribution in soleus from WT , mdx and mdx / CD38 - / - mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : E : Kolmogorov - Smirnov test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . F Images displaying the collagen ( blue ) revealed by Masson ' s trichrome staining in limb of WT , mdx , and mdx / CD38 - / - mice . The dot plot shows the quantification of collagen staining area ( % total area ) in limb of WT ( n = 4 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Scale bars : 200 µm . Data information : Each dot of the graphs represents a mouse . in triplicate . After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . G Embryonic myosin expression revealed by immunostaining along with laminin ( green ) on transverse cross - sections from limb of WT ( n = 5 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 8 ) mice . Scale bars : 50 µm . Dot plot showing the relative proportion of embryonic myosin area in limb of WT , mdx and mdx / CD38 - / - mice . Data information : Each dot of the graphs represents a mouse . one value / mouse ; After normality and variance comparison tests , significance was assessed using : G : Welch ' s ANOVA followed by Welch ' s t - tests . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A NAD+ levels in limb muscles of 20-month-old WT (n=7), mdx (n=5) and mdx/CD38-/- (n=6) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.B CD38 expression: western blot analysis of CD38 (B1) protein in limb of WT and mdx mice (n=6 per group). Vinculin is used as housekeeping protein control and the dot plots show the ratio of CD38/vinculin. qPCR analysis of CD38 mRNA (B2) in limb of 20-month-old WT and mdx mice (n=4 per group). Data information: Each dot of the graphs represents a mouse. one value/mouse; After normality and variance comparison tests, significance was assessed using: B1: unpaired Welch's t-test; B2 : unpaired Student's t-test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.C Histograms showing the grip duration (latency to fall) (C1) and the limb force (C2) measured by a grip test in WT (n=89), mdx (n=52, 53 respectively) and mdx/CD38-/- (n=58) mice (age: 9 to 26 months old). Data information: C1 one value/mouse; C2 in triplicate. After normality and variance comparison tests, significance was assessed using: : unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.D Images showing muscle fiber typology revealed by immunostaining. Localization of the slow MyHC (Type I) fiber and fast MyHCs (Type IIa and IIb/X) fibers, along with laminin (red) on transverse cross-sections from soleus and tibialis (TA) of WT, mdx and mdx/CD38-/- mice. Scale bars: 100 µm. Histogram showing the percentage of I, IIa and IIb/X fiber type distribution in soleus and TA of WT, mdx and mdx/CD38-/- mice. Experiments performed in WT (n=3), mdx (n=5) and mdx/CD38-/- (n=7) mice for soleus; WT (n=5), mdx (n=6) and mdx/CD38-/- (n=6) mice for TA. Data information: one value/mouse; After normality and variance comparison tests, significance was assessed using: D: Chi-square test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.E Fiber size distribution in soleus from WT, mdx and mdx/CD38-/- mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: E: Kolmogorov-Smirnov test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.F Images displaying the collagen (blue) revealed by Masson's trichrome staining in limb of WT, mdx, and mdx/CD38-/- mice. The dot plot shows the quantification of collagen staining area (% total area) in limb of WT (n=4), mdx (n=5) and mdx/CD38-/- (n=5) mice. Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. in triplicate. After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.G Embryonic myosin expression revealed by immunostaining along with laminin (green) on transverse cross-sections from limb of WT (n=5), mdx (n=8) and mdx/CD38-/- (n=8) mice. Scale bars: 50 µm. Dot plot showing the relative proportion of embryonic myosin area in limb of WT, mdx and mdx/CD38-/- mice. Data information: Each dot of the graphs represents a mouse. one value/mouse; After normality and variance comparison tests, significance was assessed using: G: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


Figure 6A Young mdx mice and treatment with an CD38 inhibitor . 6 - week - old mdx mice were treated for 5 weeks with K - rhein at 2 . 5 mg / kg / d by intraperitoneal injection . Dot plots showing measurement of the grip duration ( A1 ) and the force ( A2 ) of WT ( n = 9 ) , mdx ( n = 7 ) and K - rhein - treated mdx ( n = 10 ) mice . Data information : Each dot of the graphs represents a mouse . A1 one value / mouse ; A2 , in triplicate . After normality and variance comparison tests , significance was assessed using : A1 : Welch ' s ANOVA followed by Welch ' s t - tests ; A2 ANOVA followed by Fisher ' s LSD test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001B , New - born double knockout utrophin - dystrophin ( mdx / utr - / - ) mice and treatment with an CD38 inhibitor . Mdx / utr - / - mice were subcutaneously injected with K - rhein ( 0 . 6 and 2 . 5 mg / kg / d ) for 4 weeks . B : Dot plots showing the treadmill performances of K - rhein - treated mdx / utr - / - mice : distance traveled ( B1 ) , maximum speed reached ( B2 ) and maximum running time ( B3 ) ( WT ( NaCl ) and mdx / utr - / - + K - rhein 2 . 5 mg / kg / d n = 10 mice per group ; mdx / utr - / - ( NaCl ) and mdx / utr - / - + K - rhein 0 . 6 mg / kg / d , n = 12 mice per group ) . Data information : Each dot of the graphs represents a mouse . one value / mouse ; After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , # : p < 0 . 001 vs mdx / utr - / - . C New - born double knockout utrophin - dystrophin ( mdx / utr - / - ) mice and treatment with an CD38 inhibitor . Mdx / utr - / - mice were subcutaneously injected with K - rhein ( 0 . 6 and 2 . 5 mg / kg / d ) for 4 weeks . C : Measurement of the grip duration ( grid test ) in WT ( n = 10 ) , mdx / utr - / - ( n = 8 ) and K - rhein - treated mdx / utr - / - mice ( n = 10 , 6 mice , respectively for the 0 . 6 mg / kg / d and 2 . 5 mg / kg / d doses ) . Data information : Each dot of the graphs represents a mouse . in triplicate . After normality and variance comparison tests , significance was assessed using : : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , # : p < 0 . 001 vs mdx / utr - / - . D , Adult mdx mice and long - term treatment with an CD38 inhibitor . Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c ( 10 mg / kg / d ) . D : Histogram showing NAD + levels in limb of mdx ( n = 4 ) and 78c - treated mdx ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse . in duplicate ; After normality and variance comparison tests , significance was assessed using : D : unpaired Student ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001E , Adult mdx mice and long - term treatment with an CD38 inhibitor . Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c ( 10 mg / kg / d ) . E : Histogram showing grip duration ( E1 ) ( n = 8 mdx and n = 11 ) in the inverted grid test and performances in chronic treadmill test ( E2 ) at days 1 , 6 and 7 after treatment of mdx ( n = 5 excepted for D7 n = 4 ) and 78c - treated mdx ( n = 7 ) mice . Data information : Each dot of the graphs represents a mouse . in duplicate ; After normality and variance comparison tests , significance was assessed using : E1 : unpaired Mann - Whitney test ; E2 : two - way ANOVA ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001F Adult mdx mice and long - term treatment with an CD38 inhibitor . Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c ( 10 mg / kg / d ) . F : Dot plots showing barometric plethysmography measures of the tidal ( F1 ) and minute ( F2 ) volumes of mdx ( n = 4 ) and 78c - treated mdx ( n = 6 ) mice . Data information : Each dot of the graphs represents a mouse . in duplicate ; After normality and variance comparison tests , significance was assessed using : F2 , unpaired Mann - Whitney test ; F1 unpaired Welch ' s t - test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001G Time - lapse confocal imaging of calcium dynamics in human healthy and DMD myotubes loaded with the Ca2 + - sensitive dye Fluo - 4 ( white arrows show the active cells ) ( G1 ) . Traces illustrating recordings from region of interest ( ROI ) in an inactive healthy myotube ( black line ) and in a DMD myotube displaying Ca2 + spiking activity ( red line ) ( G2 ) . Histogram showing the percentage of myotubes displaying spontaneous Ca2 + waves ( G3 ) : healthy myotubes ( n = 91 cells ) , DMD myotubes ( n = 186 cells ) . Scale bars : 200 µm . Data information : After normality and variance comparison tests , significance was assessed using : G3 Chi - square test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001H Human DMD myotubes treated by SAR650984 ( isatuximab ) , a humanized anti - CD38 antibody . Fluorescence trace illustrating a recording of DMD myotubes treated with 10 µg / ml of SAR650984 ( blue ) . Histogram showing the percentage of spontaneous Ca2 + waves in DMD myotubes untreated ( n = 740 cells ) or treated with 10 µg / ml of SAR650984 ( n = 279 cells ) ( H1 ) . Histogram showing the Ca2 + wave inter - spike duration ( interval between spikes ) in myotubes treated with 10 µg / ml of SAR650984 ( n = 43 cells vs 91 for the untreated DMD myotubes ) ( H2 ) . Data information : After normality and variance comparison tests , significance was assessed using : H2 : unpaired Welch ' s t - test ; H1 : Chi - square test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Young mdx mice and treatment with an CD38 inhibitor. 6-week-old mdx mice were treated for 5 weeks with K-rhein at 2.5 mg/kg/d by intraperitoneal injection. Dot plots showing measurement of the grip duration (A1) and the force (A2) of WT (n=9), mdx (n=7) and K-rhein-treated mdx (n=10) mice. Data information: Each dot of the graphs represents a mouse. A1 one value/mouse; A2, in triplicate. After normality and variance comparison tests, significance was assessed using: A1: Welch's ANOVA followed by Welch's t-tests; A2 ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001B, New-born double knockout utrophin-dystrophin (mdx/utr-/-) mice and treatment with an CD38 inhibitor. Mdx/utr-/- mice were subcutaneously injected with K-rhein (0.6 and 2.5 mg/kg/d) for 4 weeks. B: Dot plots showing the treadmill performances of K-rhein-treated mdx/utr-/- mice: distance traveled (B1), maximum speed reached (B2) and maximum running time (B3) (WT (NaCl) and mdx/utr-/-+ K-rhein 2.5 mg/kg/d n=10 mice per group; mdx/utr-/-(NaCl) and mdx/utr-/-+ K-rhein 0.6 mg/kg/d, n=12 mice per group). Data information: Each dot of the graphs represents a mouse. one value/mouse; After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001, #: p<0.001 vs mdx/utr-/- .C New-born double knockout utrophin-dystrophin (mdx/utr-/-) mice and treatment with an CD38 inhibitor. Mdx/utr-/- mice were subcutaneously injected with K-rhein (0.6 and 2.5 mg/kg/d) for 4 weeks. C: Measurement of the grip duration (grid test) in WT (n=10), mdx/utr-/- (n=8) and K-rhein-treated mdx/utr-/- mice (n=10, 6 mice, respectively for the 0.6 mg/kg/d and 2.5 mg/kg/d doses). Data information: Each dot of the graphs represents a mouse. in triplicate. After normality and variance comparison tests, significance was assessed using: : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001, #: p<0.001 vs mdx/utr-/- .D, Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c (10 mg/kg/d). D: Histogram showing NAD+ levels in limb of mdx (n=4) and 78c-treated mdx (n=5) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: D: unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001E, Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c (10 mg/kg/d). E: Histogram showing grip duration (E1) (n=8 mdx and n=11) in the inverted grid test and performances in chronic treadmill test (E2) at days 1, 6 and 7 after treatment of mdx (n= 5 excepted for D7 n=4) and 78c-treated mdx (n= 7) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: E1: unpaired Mann-Whitney test; E2: two-way ANOVA; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001F Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c (10 mg/kg/d). F: Dot plots showing barometric plethysmography measures of the tidal (F1) and minute (F2) volumes of mdx (n=4) and 78c-treated mdx (n=6) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: F2, unpaired Mann-Whitney test ; F1 unpaired Welch's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001G Time-lapse confocal imaging of calcium dynamics in human healthy and DMD myotubes loaded with the Ca2+-sensitive dye Fluo-4 (white arrows show the active cells) (G1). Traces illustrating recordings from region of interest (ROI) in an inactive healthy myotube (black line) and in a DMD myotube displaying Ca2+ spiking activity (red line) (G2). Histogram showing the percentage of myotubes displaying spontaneous Ca2+ waves (G3): healthy myotubes (n=91 cells), DMD myotubes (n=186 cells). Scale bars: 200 µm. Data information: After normality and variance comparison tests, significance was assessed using: G3 Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001H Human DMD myotubes treated by SAR650984 (isatuximab), a humanized anti-CD38 antibody. Fluorescence trace illustrating a recording of DMD myotubes treated with 10 µg/ml of SAR650984 (blue). Histogram showing the percentage of spontaneous Ca2+ waves in DMD myotubes untreated (n=740 cells) or treated with 10 µg/ml of SAR650984 (n=279 cells) (H1). Histogram showing the Ca2+ wave inter-spike duration (interval between spikes) in myotubes treated with 10 µg/ml of SAR650984 (n=43 cells vs 91 for the untreated DMD myotubes) (H2). Data information: After normality and variance comparison tests, significance was assessed using: H2: unpaired Welch's t-test; H1: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001


Figure 1 ( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 in H1299 cells in normal medium ( HBSS ' ' - ' ' ) or after 1 hr HBSS starvation ( HBSS ' ' + ' ' ) pretreated with or without NAC ( 3mM ) . ( B ) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment . ( C ) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 ( 500μM ) treatment . CHK2 NTm , a shRNA nontargetable mutant CHK2 rescue plasmid . ( D ) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably - expressing GFP - mCherry - LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 ( 500μM ) treatment for 3h . Scale bar , 10 μm . ( E ) Quantitation of autophagosomal ( yellow ) and autolysosomal ( red ) LC3 puncta following HBSS starvation and H2O2 ( 500μM ) treatment for 1h ( n = 30 ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 ( Student ' s t - test ) . ( F ) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Scale bars , 500 nm . The red arrows indicate double - membraned autophagic structures . ( G ) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ; * * P < 0 . 01 ( Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).