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<0.001 B, 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<0.001 E, 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<0.001 F 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<0.001 G 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<0.001 H 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