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 1Whole - mount fluorescence view of E14 . 5 - 15 . 5 hearts from Npr3 - sCreER ; R26 - tdTomato and Npr3 - CreER ; R26 - tdTomato mice . Immunostaining for tdTomato , ESR and CDH5 on heart sections . C . M . , compact myocardium ; T . M . , trabecular myocardium . Arrowheads , tdTomato + ESR - endocardial cells . Quantification of the percentage of tdTomato + or ESR + endothelial cells . Data are mean ± s . e . m . ; n = 5 . Isolation of tdTomato - or tdTomato + cells from E14 . 5 Npr3 - sCreER ; R26 - tdTomato and Npr3 - CreER ; R26 - tdTomato mice by FACS . qRT - PCR of Cre , ESR , Npr3 and Cdh5 from tdTomato + cells . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Western blotting of ESR and GAPDH in tdTomato - and tdTomato + cells . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 ; n . s . , non - significant . Scale bars , 500 µm . Each figure is representative of 5 individual biological samples .

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Figure 1Whole-mount fluorescence view of E14.5-15.5 hearts from Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice. Immunostaining for tdTomato, ESR and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato+ESR- endocardial cells.Quantification of the percentage of tdTomato+ or ESR+ endothelial cells. Data are mean ± s.e.m.; n = 5.Isolation of tdTomato- or tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice by FACS.qRT-PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05.Western blotting of ESR and GAPDH in tdTomato- and tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05; n.s., non-significant. Scale bars, 500 µm. Each figure is representative of 5 individual biological samples.


Figure 2Whole - mount fluorescence images of E14 . 5 Npr3 - CreER ; R26 - Confetti and Npr3 - sCreER ; R26 - Confetti mouse hearts . No Tam is used as control for leakiness of Npr3 - sCreER . Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3 - sCreER ; R26 - Confetti heart compared with Npr3 - CreER ; R26 - Confetti heart ( Tam ) . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 5 . Scale bars , 500 µm in c ; 100 µm in d . Each image is representative of 5 individual biological samples .

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Figure 2Whole-mount fluorescence images of E14.5 Npr3-CreER;R26-Confetti and Npr3-sCreER;R26-Confetti mouse hearts. No Tam is used as control for leakiness of Npr3-sCreER.Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3-sCreER;R26-Confetti heart compared with Npr3-CreER;R26-Confetti heart (Tam). *P < 0.05; Data are mean ± s.e.m.; n = 5. Scale bars, 500 µm in c; 100 µm in d. Each image is representative of 5 individual biological samples.


Figure 3Whole mount fluorescent images of E14 . 5 or E15 . 5 Npr3 - sCreER ; R26 - LZLT and Npr3 - CreER ; R26 - LZLT mouse hearts . No tam is as control for detecting leakiness of sCreER or CreER . Representative Immunostaining images for tdTomato , ESR and CDH5 on heart sections . Quantification of the percentage of endocardial cells expressing tdTomato in Npr3 - sCreER ; R26 - LZLT ( sCreER ) or Npr3 - CreER ; R26 - LZLT ( CreER ) hearts . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm . Each figure is a representative of 5 individual biological samples .

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Figure 3Whole mount fluorescent images of E14.5 or E15.5 Npr3-sCreER;R26-LZLT and Npr3-CreER;R26-LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER. Representative Immunostaining images for tdTomato, ESR and CDH5 on heart sections.Quantification of the percentage of endocardial cells expressing tdTomato in Npr3-sCreER;R26-LZLT (sCreER) or Npr3-CreER;R26-LZLT (CreER) hearts. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm. Each figure is a representative of 5 individual biological samples.


Figure 4Immunostaining for VEGFR2 on Npr3 - sCreER ; Kdrflox / flox heart section from mice without tamoxifen treatment ( No Tam ) . Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment ( Tam ) . A , atrium ; V , ventricle ; TM , trabecular myocardium . Immunostaining for tdTomato , VEGFR2 and CDH5 on heart sections from mice of four different genotypes . Quantification data showed the percentage of VEGFR2 + endocardial cells ( ECs ) in tdTomato + ( tdT + ) cells in the TM regions . Cartoon images ( bottom ) indicate tdTomato + cells ( red ) and VEGFR2 + cells ( green ) in the TM or compact myocardium ( CM ) . Data are mean ± s . e . m . ; n = 5 . Scale bars , 100 µm . Each image is a representative of 5 individual biological samples .

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Figure 4Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam).Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.Immunostaining for tdTomato, VEGFR2 and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2+ endocardial cells (ECs) in tdTomato+ (tdT+) cells in the TM regions. Cartoon images (bottom) indicate tdTomato+ cells (red) and VEGFR2+ cells (green) in the TM or compact myocardium (CM). Data are mean ± s.e.m.; n = 5. Scale bars, 100 µm. Each image is a representative of 5 individual biological samples.


Figure 5Whole - mount fluorescence images of Col1a2 - sCreER ; R26 - tdTomato or Col1a2 - CreER ; R26 - tdTomato hearts . Inserts indicate bright - field images . Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals . Immunostaining for tdTomato and ESR on heart sections . Arrowheads indicate valve mesenchyme . Quantification of the percentage of PDGFRa + cells expressing tdTomato in Col1a2 - sCreER ; R26 - tdTomato ( sCreER ) or Col1a2 - CreER ; R26 - tdTomato ( CreER ) hearts . n . s . , non - significant ; Data are mean ± s . e . m . ; n = 6 ; n . s . , non - significant . Quantification of the percentage of tdTomato + cells expressing ESR . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 6 ; * P < 0 . 05 . Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2 - sCreER ; R26 - Confetti heart compared with Col1a2 - CreER ; R26 - Confetti heart ( Tam ) . Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm in c - e ; 100 µm in h . Each figure is a representative of 5 - 6 individual biological samples .

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Figure 5Whole-mount fluorescence images of Col1a2-sCreER;R26-tdTomato or Col1a2-CreER;R26-tdTomato hearts. Inserts indicate bright-field images.Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals.Immunostaining for tdTomato and ESR on heart sections. Arrowheads indicate valve mesenchyme. Quantification of the percentage of PDGFRa+ cells expressing tdTomato in Col1a2-sCreER;R26-tdTomato (sCreER) or Col1a2-CreER;R26-tdTomato (CreER) hearts. n.s., non-significant; Data are mean ± s.e.m.; n = 6; n.s., non-significant. Quantification of the percentage of tdTomato+ cells expressing ESR. *P < 0.05; Data are mean ± s.e.m.; n = 6; *P < 0.05.Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2-sCreER;R26-Confetti heart compared with Col1a2-CreER;R26-Confetti heart (Tam). Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm in c-e; 100 µm in h. Each figure is a representative of 5-6 individual biological samples.


Figure 1Immunoblotting of SPATC1L in testicular cells ( TC ) , testicular sperm ( TS ) , and epididymal sperm ( S ) . An anti - ADAM2 antibody was used as a control . Expression of SPATC1L in human testis lysates ( TL ) and mature sperm ( S ) . An anti - α - tubulin antibody was used as a control . Developmental expression pattern of SPATC1L in testis , examined by immunoblotting using total lysates obtained from prepubertal and adult male mice ( days 7 , 14 - 21 , 28 , 35 , 42 , 49 , and 56 ) . An anti - α - tubulin antibody was used as a control . Localization of SPATC1L in germ cells during spermatogenesis . Isolated spermatogenic cells were immunostained for SPATC1L ( red ) and γ - tubulin ( green ) . γ - Tubulin was used as a centrosome marker , and cells were counterstained with the nuclear dye Hoechst 33342 ( blue ) . Fluorescence images of individual staining are shown at the top and merged images are shown in the middle . Insets are magnified iamges . Sc , spermatocyte ; RS , round spermatid ; ES , elongating spermatid ; TS , testicular sperm ; MS , epididymal mature sperm ; BF , overlay with bright field image .

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Figure 1Immunoblotting of SPATC1L in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). An anti-ADAM2 antibody was used as a control.Expression of SPATC1L in human testis lysates (TL) and mature sperm (S). An anti-α-tubulin antibody was used as a control.Developmental expression pattern of SPATC1L in testis, examined by immunoblotting using total lysates obtained from prepubertal and adult male mice (days 7, 14-21, 28, 35, 42, 49, and 56). An anti-α-tubulin antibody was used as a control.Localization of SPATC1L in germ cells during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and γ-tubulin (green). γ-Tubulin was used as a centrosome marker, and cells were counterstained with the nuclear dye Hoechst 33342 (blue). Fluorescence images of individual staining are shown at the top and merged images are shown in the middle. Insets are magnified iamges. Sc, spermatocyte; RS, round spermatid; ES, elongating spermatid; TS, testicular sperm; MS, epididymal mature sperm; BF, overlay with bright field image.


Figure 2Immunoprecipitation of SPATC1L in the testis using an anti - SPATC1L antibody . IgG was used as a control . In , input ; IgG , normal rabbit serum . Interaction between SPATC1L and PKA subunits . Samples precipitated with an anti - SPATC1L antibody were analyzed by immunoblotting with anti - RIα and anti - Cα antibodies . Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti - SPATC1L antibody . IgG was used as a control . IP , immunoprecipitation ; In , input ; IgG , immunoglobulin G from normal rabbit serum . Expression of PKA subunits during spermatogenesis , examined by immunoblotting for RIα and Cα in testicular cells ( TC ) , testicular sperm ( TS ) , and epididymal sperm ( S ) . Anti - ADAM2 and anti - α - tubulin antibodies were used as controls . Localization of SPATC1L and RIα during spermatogenesis . Isolated spermatogenic cells were immunostained for SPATC1L ( red ) and RIα ( green ) , and counterstained with the nuclear dye Hoechst 33342 ( blue ) . Overlay with bright field image is shown at right in each cell type . TC , testicular cell ( spermatocyte ) ; RS , round spermatid ; ES , elongating spermatid ; TS , testicular sperm . Scale bar = 5 μm .

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Figure 2Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum.Interaction between SPATC1L and PKA subunits. Samples precipitated with an anti-SPATC1L antibody were analyzed by immunoblotting with anti-RIα and anti-Cα antibodies. Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti-SPATC1L antibody. IgG was used as a control. IP, immunoprecipitation; In, input; IgG, immunoglobulin G from normal rabbit serum.Expression of PKA subunits during spermatogenesis, examined by immunoblotting for RIα and Cα in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). Anti-ADAM2 and anti-α-tubulin antibodies were used as controls.Localization of SPATC1L and RIα during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and RIα (green), and counterstained with the nuclear dye Hoechst 33342 (blue). Overlay with bright field image is shown at right in each cell type. TC, testicular cell (spermatocyte); RS, round spermatid; ES, elongating spermatid; TS, testicular sperm. Scale bar = 5 μm.


Figure 3Mapping of the SPATC1L binding site on RIα by in vitro binding assay using purified GST - SPATC1L and His - RIα proteins consisting of regions 1 - 6 . Purified GST protein only was used as a control . Binding was confirmed by immunoprecipitation using an anti - SPATC1L antibody followed by immunoblot analyses of RIα , detected using an anti - His antibody . In , input ; Sup , supernatant ; IP , immunoprecipitated protein . PKA activity after overexpression of SPATC1L or SPATC1LΔC . Graphs show the average PKA activity of Spatc1l - transfected cells ( white bar ) and Spatc1lΔC - transfected cells ( black bar ) in the presence or absence of cAMP , expressed relative to control cells ( mock ) as a percentage . Representative signals observed in the assay are shown above . The overexpression of SPATC1L was confirmed in every experiment by immunoblotting ( data not shown ) . The amount of Cα associated with RIα in the presence or absence of SPATC1LΔC . After IP of RIα in cells overexpressing SPATC1LΔC or control , immunoblot analysis was performed using anti - Cα antibody . Graph shows densitometric analysis of Cα in relation to immunoprecipitated RIα in immunoblot analysis . In , input ; IgG , immunoglobulin G from normal rabbit serum .

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Figure 3Mapping of the SPATC1L binding site on RIα by in vitro binding assay using purified GST-SPATC1L and His-RIα proteins consisting of regions 1-6. Purified GST protein only was used as a control. Binding was confirmed by immunoprecipitation using an anti-SPATC1L antibody followed by immunoblot analyses of RIα, detected using an anti-His antibody. In, input; Sup, supernatant; IP, immunoprecipitated protein.PKA activity after overexpression of SPATC1L or SPATC1LΔC. Graphs show the average PKA activity of Spatc1l-transfected cells (white bar) and Spatc1lΔC-transfected cells (black bar) in the presence or absence of cAMP, expressed relative to control cells (mock) as a percentage. Representative signals observed in the assay are shown above. The overexpression of SPATC1L was confirmed in every experiment by immunoblotting (data not shown).The amount of Cα associated with RIα in the presence or absence of SPATC1LΔC. After IP of RIα in cells overexpressing SPATC1LΔC or control, immunoblot analysis was performed using anti-Cα antibody. Graph shows densitometric analysis of Cα in relation to immunoprecipitated RIα in immunoblot analysis . In, input; IgG, immunoglobulin G from normal rabbit serum.


Figure 4PCR genotyping analysis of WT ( Spatc1l + / + ) , heterozygous ( Spatc1l + / - ) and homozygous ( Spatc1l - / - ) mice . SPATC1L expression levels in testes obtained from WT , Spatc1l + / - , and Spatc1l - / - mice . An anti - GAPDH antibody was used as a control . Graph shows densitometric analysis of SPATC1L in relation to GAPDH in immunoblot analysis . Percentage of females that became pregnant after mating with WT ( white bar ) , Spatc1l + / - ( gray bar ) , and Spatc1l - / - ( black bar ) male mice . Male mice used : WT , n = 5 ; Spatc1l + / - , n = 6 ; Spatc1l - / - , n = 7 . WT female mice used for mating with the indicated genotype : WT , n = 30 ; Spatc1l + / - , n = 36 ; Spatc1l - / - , n = 42 . Vaginal plugs were observed in all mated females . Average litter size of pups produced by females mated with WT ( white bar ) , Spatc1l + / - ( gray bar ) , and Spatc1l - / - ( black bar ) male mice .

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Figure 4PCR genotyping analysis of WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice.SPATC1L expression levels in testes obtained from WT, Spatc1l+/-, and Spatc1l-/- mice. An anti-GAPDH antibody was used as a control. Graph shows densitometric analysis of SPATC1L in relation to GAPDH in immunoblot analysis.Percentage of females that became pregnant after mating with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice. Male mice used: WT, n = 5; Spatc1l+/-, n = 6; Spatc1l-/-, n = 7. WT female mice used for mating with the indicated genotype: WT, n = 30; Spatc1l+/-, n = 36; Spatc1l-/-, n = 42. Vaginal plugs were observed in all mated females.Average litter size of pups produced by females mated with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice.


Figure 5Macroscopic appearance of adult testes from an 8 - week - old WT ( Spatc1l + / + ) , heterozygous ( Spatc1l + / - ) and homozygous ( Spatc1l - / - ) mice . Scale bar = 2 mm . Comparison of testis weight in WT ( white bar ) , Spatc1l + / - ( gray bar ) , and Spatc1l - / - ( black bar ) mice . The organs were trimmed of fat and weighed . The average testis weight / body weight ratios for WT , Spatc1l + / - , and Spatc1l - / - males were 0 . 0034 ± 0 . 0001 , 0 . 0038 ± 0 . 0003 , and 0 . 0031 ± 0 . 0002 , respectively . Data are presented as means ± s . d . ( n = 7 ) . Macroscopic appearance of the adult epididymis from WT , Spatc1l + / - , and Spatc1l - / - mice . Scale bar = 2 mm . Epididymis weight in WT ( white bar ) , Spatc1l + / - ( gray bar ) , and Spatc1l - / - ( black bar ) mice . The average epididymis weight / body weight ratios for WT , Spatc1l + / - , and Spatc1l - / - males were 0 . 001 ± 0 . 0001 , 0 . 001 ± 0 . 0001 , and 0 . 001 ± 0 . 00008 , respectively . Data are presented as means ± s . d . ( n = 7 ) . Histological analysis of hematoxylin and eosin - stained sections of seminiferous tubules prepared from testes of WT and KO mice . Scale bar = 100 μm ( upper ) and 50 μm ( lower ) . Number of mature epididymal sperm from WT ( white bar ) , Spatc1l + / - ( gray bar ) , and Spatc1l - / - ( black bar ) mice . Sperm were collected from the vas deferens . The average number of mature sperm in WT , Spatc1l + / - , and Spatc1l - / - males was ( 4 . 06 ± 0 . 40 ) × 106 , ( 3 . 7 ± 0 . 7 ) × 106 , and ( 3 . 93 ± 0 . 90 ) × 106 , respectively . Data are presented as means ± s . d . ( n = 5 ) . Morphological analyses of epididymal sperm in WT , He and KO mice . Epididymal sperm were observed under an inverted microscope . The arrows indicate separated tails , black arrowheads indicate separated heads and white arrowheads indicate folded heads . Scale bar = 50 μm . Percentage of headless sperm from cauda epididymides of WT ( black ) , Spatc1l + / - ( gray ) , and Spatc1l - / - ( white ) mice . The average percentage of headless sperm in WT , Spatc1l + / - , and Spatc1l - / - males was 6 . 3 , 52 . 9 and 100 , respectively . Each dot represents percentage of headless sperm in an individual mouse , and medians are represented as horizontal lines . ( n = 13 , * P < 0 . 001 , Student ' s t - test ) . Immunostaining analysis of sperm heads and tails in WT and KO mice . Sperm tails were observed by immunostaining for α - tubulin antibody ( red ) , and counterstaining with the nuclear dye Hoechst 33342 ( blue ) . Sperm were also observed by differential interference contrast ( DIC ) microscopy . Scale bars = 20 μm ( upper ) and 5 μm ( lower ) .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O 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

Figure 5Macroscopic appearance of adult testes from an 8-week-old WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice. Scale bar = 2 mm.Comparison of testis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The organs were trimmed of fat and weighed. The average testis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.0034±0.0001, 0.0038±0.0003, and 0.0031±0.0002, respectively. Data are presented as means ± s.d. (n = 7).Macroscopic appearance of the adult epididymis from WT, Spatc1l+/-, and Spatc1l-/- mice. Scale bar = 2 mm.Epididymis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The average epididymis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.001±0.0001, 0.001±0.0001, and 0.001±0.00008, respectively. Data are presented as means ± s.d. (n = 7).Histological analysis of hematoxylin and eosin-stained sections of seminiferous tubules prepared from testes of WT and KO mice. Scale bar = 100 μm (upper) and 50 μm (lower).Number of mature epididymal sperm from WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. Sperm were collected from the vas deferens. The average number of mature sperm in WT, Spatc1l+/-, and Spatc1l-/- males was (4.06 ± 0.40) × 106, (3.7 ± 0.7) × 106, and (3.93 ± 0.90) × 106, respectively. Data are presented as means ± s.d. (n = 5).Morphological analyses of epididymal sperm in WT, He and KO mice. Epididymal sperm were observed under an inverted microscope. The arrows indicate separated tails, black arrowheads indicate separated heads and white arrowheads indicate folded heads. Scale bar = 50 μm.Percentage of headless sperm from cauda epididymides of WT (black), Spatc1l+/- (gray), and Spatc1l-/- (white) mice. The average percentage of headless sperm in WT, Spatc1l+/-, and Spatc1l-/- males was 6.3, 52.9 and 100, respectively. Each dot represents percentage of headless sperm in an individual mouse, and medians are represented as horizontal lines. (n=13, *P < 0.001, Student's t-test).Immunostaining analysis of sperm heads and tails in WT and KO mice. Sperm tails were observed by immunostaining for α-tubulin antibody (red), and counterstaining with the nuclear dye Hoechst 33342 (blue). Sperm were also observed by differential interference contrast (DIC) microscopy. Scale bars = 20 μm (upper) and 5 μm (lower).


Figure 6Morphological analyses of Spatc1l - KO epididymal sperm by SEM . Sperm were obtained from the vas deferens of 8 - week - old WT ( Spatc1l + / + ) and KO ( Spatc1l - / - ) mice . Sperm neck regions are magnified in lower panels . AC , acrosomal cap ; MP , mid piece ; BP , basal plates . Scale bar = 2 μm ( upper ) and 0 . 5 μm ( lower ) . TEM analyses of flagella in WT and KO testicular sperm . TEM images represent ultrastructures of cross - sections of the midpiece ( upper ) and end piece ( lower ) of WT and KO testicular sperm . Ax , axonemal microtubules ; Mt , mitochondrial sheath ; ODF , outer dense fibers . Scale bars = 200 nm ( upper ) and 100 nm ( lower ) . TEM analyses of the development of the connecting piece in WT ( left ) and KO ( right ) sperm . TEM images represent ultrastructures of longitudinal sections of elongating spermatids ( ES ) and testicular sperm ( TS ) in WT and KO mice . An , annulus ; BP , basal plates ; Cp , capitulum ; Mc , manchette ; Mt , mitochondria ; Nu , nucleus ; PCV , proximal centriolar vault ; PM , plasma membrane ; SC , segmented column . Scale bar = 500 nm . Ultrastructures of epididymal sperm in KO mice . TEM images represent ultrastructures of separated sperm head ( upper ) and tail ( lower ) in the epididymis of KO mice . Images in the boxes are enlarged at right . BP , basal plates ; Cp , capitulum ; SC , segmented column . Scale bar = 500 nm . TEM analysis of morphologically abnormal KO sperm . Detached heads abnormally located to the midpiece of sperm flagella were observed by TEM ( left ) and confocal differential interference contrast ( DIC ) microscopy ( right ) . PM , plasma membrane . Scale bars = 500 nm ( left ) and 5 μm ( right ) .

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Figure 6Morphological analyses of Spatc1l-KO epididymal sperm by SEM. Sperm were obtained from the vas deferens of 8-week-old WT (Spatc1l+/+) and KO (Spatc1l-/-) mice. Sperm neck regions are magnified in lower panels. AC, acrosomal cap; MP, mid piece; BP, basal plates. Scale bar = 2 μm (upper) and 0.5 μm (lower).TEM analyses of flagella in WT and KO testicular sperm. TEM images represent ultrastructures of cross-sections of the midpiece (upper) and end piece (lower) of WT and KO testicular sperm. Ax, axonemal microtubules; Mt, mitochondrial sheath; ODF, outer dense fibers. Scale bars = 200 nm (upper) and 100 nm (lower).TEM analyses of the development of the connecting piece in WT (left) and KO (right) sperm. TEM images represent ultrastructures of longitudinal sections of elongating spermatids (ES) and testicular sperm (TS) in WT and KO mice. An, annulus; BP, basal plates; Cp, capitulum; Mc, manchette; Mt, mitochondria; Nu, nucleus; PCV, proximal centriolar vault; PM, plasma membrane; SC, segmented column. Scale bar = 500 nm.Ultrastructures of epididymal sperm in KO mice. TEM images represent ultrastructures of separated sperm head (upper) and tail (lower) in the epididymis of KO mice. Images in the boxes are enlarged at right. BP, basal plates; Cp, capitulum; SC, segmented column. Scale bar = 500 nm.TEM analysis of morphologically abnormal KO sperm. Detached heads abnormally located to the midpiece of sperm flagella were observed by TEM (left) and confocal differential interference contrast (DIC) microscopy (right). PM, plasma membrane. Scale bars = 500 nm (left) and 5 μm (right).


Figure 7PKA activities in testicular sperm from WT ( + / + ) and KO ( - / - ) mice . Graph shows average PKA activity in testicular sperm from KO mice ( black bar ) relative to that from WT mice ( white bar ) , expressed as a percentage . Representative ProQ Diamond - stained 2D gel images of isolated proteins from testes in WT ( left ) and KO ( right ) mice . Testes lysates were separated by 2DE , and phosphoproteins were stained with ProQ diamond . Images in the boxes are enlarged in insets shown below . Circles with numbers indicate spots with significantly changed volume relative to those on WT gels . Comparative phosphorylation intensities of selected spots in 2D gels . The intensities of selected spots in KO gels were compared with intensities in WT gels . Average intensities of the indicated spot numbers ( # ) in WT ( white bar ) and KO ( black bar ) : # 1205 , 1092 . 2 ( WT ) and 244 . 98 ( KO ) ; # 1207 , 729 . 9 ( WT ) and 140 . 43 ( KO ) ; # 3303 , 1165 . 5 ( WT ) and 279 . 5 ( KO ) ; # 4302 , 2725 . 8 ( WT ) and 1 ( KO ) ; # 4405 , 984 . 5 ( WT ) and 1 ( KO ) . An intensity value of 1 indicates no spot , but is provided for calculation of fold change . Immunoblot analyses of five candidate proteins in testis ( E ) and sperm ( F ) of WT and KO mice . Anti - GAPDH , anti - ADAM2 , and anti - α - tubulin antibodies were used as controls . Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti - SPATC1L antibody . Localization of SPATC1L ( red ) and CAPZB ( green ) in testicular sperm . Overlay with bright field image is shown at right . The white arrow indicates the spatial overlap between SPATC1L and CAPZB . Scale bar = 5 μm .

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Figure 7PKA activities in testicular sperm from WT (+/+) and KO (-/-) mice. Graph shows average PKA activity in testicular sperm from KO mice (black bar) relative to that from WT mice (white bar), expressed as a percentage.Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels.Comparative phosphorylation intensities of selected spots in 2D gels. The intensities of selected spots in KO gels were compared with intensities in WT gels. Average intensities of the indicated spot numbers (#) in WT (white bar) and KO (black bar): #1205, 1092.2 (WT) and 244.98 (KO); #1207, 729.9 (WT) and 140.43 (KO); #3303, 1165.5 (WT) and 279.5 (KO); #4302, 2725.8 (WT) and 1 (KO); #4405, 984.5 (WT) and 1 (KO). An intensity value of 1 indicates no spot, but is provided for calculation of fold change.Immunoblot analyses of five candidate proteins in testis (E) and sperm (F) of WT and KO mice. Anti-GAPDH, anti-ADAM2, and anti-α-tubulin antibodies were used as controls. Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti-SPATC1L antibody.Localization of SPATC1L (red) and CAPZB (green) in testicular sperm. Overlay with bright field image is shown at right. The white arrow indicates the spatial overlap between SPATC1L and CAPZB. Scale bar = 5 μm.


Figure 8The amount of CAPZB in mature sperm from WT ( + / + ) and KO ( - / - ) mice . Graph shows densitometric analysis of CAPZB shown in immunoblot analyses ( Figure 7F ) , expressed relative to WT as a percentage . The density of CAPZB in immunoblot analysis was calculated in relation to density of α - Tubulin . The amount of CAPZB in mock and Spatc1lΔC - transfected cells . Anti - GAPDH antibody was used as control . Overexpression of SPATC1L in TM4 cells was confirmed by immunoblotting with anti - SPATC1L antibody . Graph shows densitometric analysis of CAPZB in pcDNA3 . 1 vector - transfected control cells ( mock ; white bar ) and SPATC1LΔC - transfected cells ( black bar ) , expressed relative to control cells ( mock ) as a percentage . The density of CAPZB in immunoblot analysis was calculated in relation to density of GAPDH . Phosphorylation of CPAZB3 by cAMP - dependent protein kinase ( PKA ) . CAPZB3 was incubated with PKA and 32P , and estimated by autoradiography . The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue ( CBB ) staining ( lower ) . Phosphorylation of CPAZB3 in the presence or absence of SPATC1L . Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L , and estimated by autoradiography . The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue ( CBB ) staining ( lower ) . Graph shows densitometric analysis of phopho - CAPZB3 ( p - CAPZB3 ) in reactions without ( control ; white bar ) or with SPATC1L ( black bar ) , expressed relative to control as a percentage . Observation of filamentous actin ( F - actin ) in the presence ( lower ) or absence ( upper ) of SPATC1L . F - actin in pEGFP vector - transfected control cells ( mock ; upper , green ) and cells overexpressing SPATC1LΔC ( lower , green ) was visualized by staining using Phalloidin ( red ) . White bar shows the length of transfected cell . Scale bar = 50 μm . The length of cell overexpressing SPATC1LΔC compared with control . Graph shows the average of measured cell length in pEGFP vector - transfected control cells ( mock ; white bar ) and Spatc1lΔC - transfected cells ( black bar ) , expressed relative to control cells ( mock ) as a percentage .

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Figure 8The amount of CAPZB in mature sperm from WT (+/+) and KO (-/-) mice. Graph shows densitometric analysis of CAPZB shown in immunoblot analyses (Figure 7F), expressed relative to WT as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of α-Tubulin.The amount of CAPZB in mock and Spatc1lΔC-transfected cells. Anti-GAPDH antibody was used as control. Overexpression of SPATC1L in TM4 cells was confirmed by immunoblotting with anti-SPATC1L antibody. Graph shows densitometric analysis of CAPZB in pcDNA3.1 vector-transfected control cells (mock; white bar) and SPATC1LΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of GAPDH.Phosphorylation of CPAZB3 by cAMP-dependent protein kinase (PKA). CAPZB3 was incubated with PKA and 32P, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower).Phosphorylation of CPAZB3 in the presence or absence of SPATC1L. Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Graph shows densitometric analysis of phopho-CAPZB3 (p-CAPZB3) in reactions without (control; white bar) or with SPATC1L (black bar), expressed relative to control as a percentage.Observation of filamentous actin (F-actin) in the presence (lower) or absence (upper) of SPATC1L. F-actin in pEGFP vector-transfected control cells (mock; upper, green) and cells overexpressing SPATC1LΔC (lower, green) was visualized by staining using Phalloidin (red). White bar shows the length of transfected cell. Scale bar = 50 μm.The length of cell overexpressing SPATC1LΔC compared with control. Graph shows the average of measured cell length in pEGFP vector-transfected control cells (mock; white bar) and Spatc1lΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage.


figf1 ( a - f ) Muscle biopsy from subject 4 . 1 subjected to electron microscopy ( transverse sections ) . In many fibers , there is material between the layers of basal lamina ( a , arrows ; scale bar , 500 nm ) or overt exocytic vacuoles ( b , arrow ; scale bar , 2 μm ) . Some fibers show a single centralized nucleus ( c ; scale bar , 2 μm ) . Mitochondria are of variable size and distribution . In some fibers , they form a loop around the nucleus in the periphery of the fiber , resembling a necklace as in c ; in others , they form clusters ( d ; scale bar , 2 μm ) . The appearance of cristae is often abnormal ( e ; scale bar , 2 μm ; f ; scale bar , 500 nm ) . N , nucleus ; M , mitochondrion .

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figf1(a-f) Muscle biopsy from subject 4.1 subjected to electron microscopy (transverse sections). In many fibers, there is material between the layers of basal lamina (a, arrows; scale bar, 500 nm) or overt exocytic vacuoles (b, arrow; scale bar, 2 μm). Some fibers show a single centralized nucleus (c; scale bar, 2 μm). Mitochondria are of variable size and distribution. In some fibers, they form a loop around the nucleus in the periphery of the fiber, resembling a necklace as in c; in others, they form clusters (d; scale bar, 2 μm). The appearance of cristae is often abnormal (e; scale bar, 2 μm; f; scale bar, 500 nm). N, nucleus; M, mitochondrion.


figf2Transverse sections from muscle biopsies from subject 3 . 1 ( top ) and a normal control ( bottom ) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M - band titin , titin M8 . Accumulation of NBR1 in puncta ( autophagosomes , arrowheads ) and of MURF2 , as well as fiber inhomogeneity with marked fiber atrophy , characterizes Vici syndrome muscle . Numerous fibers of very small cross - sectional area ( arrows ) with high content of MURF2 and NBR1 puncta are frequently seen . Scale bar , 10 μm .

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figf2Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.


figf3Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome ( subject 4 . 1 ) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin .

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figf3Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome (subject 4.1) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin.


figf4In control fibroblasts subjected to 6 h of bafilomycin treatment , lysosomal structures were detected by staining with monoclonal antibody to LAMP1 . Numerous LC3 - positive autophagosomes are found engulfed by the LAMP1 - positive vesicular structures ( arrowheads ) . In contrast , fibroblasts from individuals with Vici syndrome consistently show smaller LC3 - positive puncta that only sporadically colocalize with LAMP1 , with many isolated LC3 - positive puncta ( arrows ) . Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1 - positive structures , not centrally . Scale bar , 5 μm .

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figf4In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal structures were detected by staining with monoclonal antibody to LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, fibroblasts from individuals with Vici syndrome consistently show smaller LC3-positive puncta that only sporadically colocalize with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar, 5 μm.


Figure 1qRT - PCR analysis of Foxp1 - 4 in murine ECs freshly isolated from P11 mesentery ( n = 6 mice , individual data points shown ) . Data are presented as mean relative expression ( normalized to Gapdh ) ± SD . Transcript levels for each transcript are presented relative to levels in LECs . P , Student ' s t - test . nd , not detected .

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Figure 1qRT-PCR analysis of Foxp1-4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected.


Figure 2Immunofluorescence ( C ) and qRT - PCR analysis ( D ) of HDLECs grown under static conditions or exposed to OSS for 48 h ( n = 3 independent experiments ) . Data are presented as mean ± SD . P , Student ' s t - test .

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Figure 2Immunofluorescence (C) and qRT-PCR analysis (D) of HDLECs grown under static conditions or exposed to OSS for 48 h (n = 3 independent experiments). Data are presented as mean ± SD. P, Student's t-test.


Figure 3Whole - mount immunofluorescence of P7 mesenteries , showing efficient FOXP2 depletion in Foxp2flox / - ; Tie2 - Cre vessels . Dotted line indicates vessel width . Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox / - ; Tie2 - Cre and littermate controls ( n = vessels [ mice ] , as indicated ) . Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox / - ; Tie2 - Cre mice . A spectrum of defects in the mutants ranging from shortened integrin - α9 + FN - EIIIA + valve leaflets ( middle panels ) to a complete loss of leaflets ( right panels ) are shown . Quantification of the proportion of valves with integrin - α9 + leaflets ( G ) , and leaflet length ( H ) in P7 Foxp2flox / - ; Tie2 - Cre and littermate controls ( n = valves [ mice ] , as indicated ) . Leaflet length = 0 corresponds to valves with no leaflets .

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Figure 3Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/-;Tie2-Cre vessels. Dotted line indicates vessel width. Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = vessels [mice], as indicated).Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/-;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown. Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets.


Figure 4Whole - mount immunofluorescence ( A ) and quantification of NRP2 staining intensity ( B ) in P6 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre and littermate control mice ( n = vessels [ mice ] , as indicated ) . RNAscope - based whole - mount in situ hybridization ( C ) and quantification of Efnb2 transcript levels ( D ) in P7 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre and littermate control mice ( n = vessels [ mice ] , as indicated ) .

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Figure 4Whole-mount immunofluorescence (A) and quantification of NRP2 staining intensity (B) in P6 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).


Figure 5Whole - mount immunofluorescence of P6 mesenteries , showing valve defects in the mutant vessels . Quantification of lymphatic valve numbers ( C ) , valve leaflet length ( D ) and vessel width ( E ) in Foxp2flox / flox ; Prox1 - CreERT2 and littermate control mice ( n = vessels ( C , E ) or valves ( D ) [ mice ] , as indicated ) .

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Figure 5Whole-mount immunofluorescence of P6 mesenteries, showing valve defects in the mutant vessels. Quantification of lymphatic valve numbers (C), valve leaflet length (D) and vessel width (E) in Foxp2flox/flox;Prox1-CreERT2 and littermate control mice (n = vessels (C, E) or valves (D) [mice], as indicated).


Figure 6qRT - PCR analysis of FOXP2 , FOXC2 and GJA4 expression in control ( siCTRL ) and FOXP2 or FOXC2 siRNA - treated HDLECs ( n = 6 ( FOXP2 , FOXC2 ) or n = 3 ( GJA4 ) independent experiments ) . Top panels : whole - mount immunofluorescence of E18 Foxc2flox / flox ; Prox1 - CreERT2 and littermate control mesenteries , showing downregulation of FOXP2 in Foxc2 deficient vessels . Bottom panels : extraction of nuclear FOXP2 staining ( grey ) using IMARIS surface mask generated based on PROX1 staining ( green ) . Unmasked FOXP2 staining is shown in magenta . Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox / flox ; Prox1 - CreERT2 and control littermates based on nuclear FOXP2 signals as in ( B ) ( n = vessels [ mice ] , as indicated ) . Whole - mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels . Whole - mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox / - ; Tie2 - Cre mice showing NFATc1 subcellular localization . Note predominantly nuclear localization of NFATc1 in control valves ( left panels ) , but cytoplasmic localization ( middle panels , 3 out of 5 mice ) or downregulation of NFATc1 ( right panels , 2 out of 5 mice ) in the mutant valves . Quantification of protein localization in NFATc1 + lymphatic valves from ( D ) ( n = valves [ mice ] , as indicated ) .

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Figure 6qRT-PCR analysis of FOXP2, FOXC2 and GJA4 expression in control (siCTRL) and FOXP2 or FOXC2 siRNA-treated HDLECs (n = 6 (FOXP2, FOXC2) or n = 3 (GJA4) independent experiments).Top panels: whole-mount immunofluorescence of E18 Foxc2flox/flox;Prox1-CreERT2 and littermate control mesenteries, showing downregulation of FOXP2 in Foxc2 deficient vessels. Bottom panels: extraction of nuclear FOXP2 staining (grey) using IMARIS surface mask generated based on PROX1 staining (green). Unmasked FOXP2 staining is shown in magenta. Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox/flox;Prox1-CreERT2 and control littermates based on nuclear FOXP2 signals as in (B) (n = vessels [mice], as indicated).Whole-mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels. Whole-mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox/-;Tie2-Cre mice showing NFATc1 subcellular localization. Note predominantly nuclear localization of NFATc1 in control valves (left panels), but cytoplasmic localization (middle panels, 3 out of 5 mice) or downregulation of NFATc1 (right panels, 2 out of 5 mice) in the mutant valves. Quantification of protein localization in NFATc1+ lymphatic valves from (D) (n = valves [mice], as indicated).


Figure 1Overexpression of WT OGT , but not catalytic - inactive OGT mutant , increased the O - GlcNAc levels of TDP - 43 . OGT protein levels were examined by immunoblotting . Quantification of ( G ) .

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Figure 1Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).


Figure 2Yeast cells expressing the indicated constructs were induced in galactose - containing medium , and representative images were shown using fluorescence microscopy to visualize TDP - 43 aggregates . DIC : Differential interference contrast . TDP - 43 aggregate foci numbers from individual cell of ( B ) were quantified from over 50 cells each of the indicated yeast strains . The foci numbers in individual cell > 3 were labelled with yellow , and the foci numbers in individual cell < 3 are labelled with blue .

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Figure 2Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue.


Figure 3SH - SY5Y cells treated with the indicated compounds were immunostained with TDP - 43 or pTDP - 43 and observed by immunofluorescence . Quantification of signal intensities of pTDP - 43 staining ( left ) or foci numbers per cell ( right ) . Cell number counted : left panel , " Mock " n = 58 , " GlcNAc " n = 35 , " EA " n = 45 , " GlcNAc + EA " n = 52 ; right panel , " Mock " n = 1167 , " GlcNAc " n = 1051 , " EA " n = 542 , " GlcNAc + EA " n = 815 . SDS - soluble and insoluble fractions were isolated from SH - SY5Y cells expressing WT or catalytic - inactive mutant OGT treated with 20 μM EA , and the fractions were analyzed by immunoblotting . Quantification of ( F ) .

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Figure 3SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815.SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F).


Figure 4Cells expressing the indicated forms of GFP - TDP - 43 were subjected to immunoprecipitation , and the O - GlcNAcylated levels of TDP - 43 were examined . Quantification of ( G ) .

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Figure 4Cells expressing the indicated forms of GFP-TDP-43 were subjected to immunoprecipitation, and the O-GlcNAcylated levels of TDP-43 were examined.   Quantification of (G).


Figure 5Walking speed of 10 - day old adult male flies of the indicated genotypes was calculated Mean ± SD . Unpaired two - tailed t - test , biological replicates : Control , n = 63 , WT , n = 45 , T199A , n = 70 , T233A , n = 87 ; 2TA , n = 43 . . Adult flies were fed with regular sucrose diet ( RS ) or high sucrose diet ( HS ) , and survival percentage of adult flies of the indicated genotypes were examined . Kaplan - Meier survival analysis , n ≥ 40 for each group .

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Figure 5Walking speed of 10-day old adult male flies of the indicated genotypes was calculated Mean ± SD. Unpaired two-tailed t-test, biological replicates: Control, n = 63, WT, n = 45, T199A, n = 70, T233A, n = 87; 2TA, n = 43.   .Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.


Figure 6CFTR splicing assay was performed in cells with knockdown of either OGT , TDP - 43 , or combination . ACTB levels served as loading controls ( top panel ) . The indicated protein levels were examined by immunoblotting ( bottom panel ) . CFTR splicing assay was examined in siTDP - 43 cells overexpressing the WT or mutant OGT by PCR analysis . ACTB levels served as loading controls ( top panel ) . Various protein levels were examined by immunoblotting ( bottom panel ) . Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH - SY5Y cells with the indicated constructs ( top panel ) . TDP - 43 protein levels were examined by immunoblotting ( bottom panel ) .

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Figure 6CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).


Figure 1 ( B ) Representative images of mCherry - optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in ( A ) . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) . ( C ) Representative images of the same cells in ( B ) exposed to both 488 and 561 nm following the protocol shown in ( A ) . Scale bars , 10 μm . ( C ' ) Fold - change in nuclear localisation of the mCherry - optoYAP ( n = 50 cells from 3 independent experiments ) . ( D ) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark . ( Top ) The ratio of mCherry - optoYAP signal in the nucleus to the cytoplasmic mCherry - optoYAP signal ( n = 8 cells from 2 independent experiments ) . ( Bottom ) Representative images of the mCherry - optoYAP signal from the same HEK293T cells at each cycle . Scale bars , 10 μm .

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Figure 1(B) Representative images of mCherry-optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in (A). Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optoYAP (n=50 cells from 3 independent experiments).(C) Representative images of the same cells in (B) exposed to both 488 and 561 nm following the protocol shown in (A). Scale bars, 10 μm. (C') Fold-change in nuclear localisation of the mCherry-optoYAP (n=50 cells from 3 independent experiments).(D) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark. (Top) The ratio of mCherry-optoYAP signal in the nucleus to the cytoplasmic mCherry-optoYAP signal (n=8 cells from 2 independent experiments). (Bottom) Representative images of the mCherry-optoYAP signal from the same HEK293T cells at each cycle. Scale bars, 10 μm.


Figure 3 ( B ) Example images of embryos expressing optofYap ( mCherry - tagged ) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf . Scale bars , 10 μm . ( B ' ) Fold - change in nuclear localisation of mCherry - optofYap ( n = 20 cells from 3 independent experiments ) .

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Figure 3(B) Example images of embryos expressing optofYap (mCherry-tagged) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf. Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optofYap (n=20 cells from 3 independent experiments).


Figure 4Soft agar colony formation assay for HFF ( A - B ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in HFF cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates . Soft agar colony formation assay for MKN28 ( C - D ) cells respectively grown on soft agar for one week under different light conditions as described in Fig . 1A . Scale bars , 1 mm . Representative images of colony growth in MKN28 cells respectively under different activation conditions . Diameter of individual colonies formed under different light conditions and genetic backgrounds . Colonies were counted and measured from 3 biological replicates .

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Figure 4Soft agar colony formation assay for HFF (A-B) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in HFF cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.Soft agar colony formation assay for MKN28 (C-D) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in MKN28 cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


Figure 1 ( B ) Example images of brightfield and fluorescently labelled HPG ( Alexa Fluor 647 ) of wild type cells ( PN1 ) assayed for global cellular translation . Scale bars represent 5 µm . ( C ) Change in HPG labelling signal with different durations of HPG incubation ( in PN1 ) , measured by flow cytometry . Population medians of at least 200 , 000 cells are shown . The red line is the ordinary least square ( OLS ) linear regression fitted on the medians between 0 and 5 minutes . ( ( E ) Example images of brightfield and fluorescently labelled EU ( Alexa Fluor 488 ) of hENT1 and hsvTK cells ( PN10567 ) assayed for global cellular transcription . Scale bars represent 5 µm . ( F ) Change in EU labelling signal with different lengths of EU incubations , measured by flow cytometry ( in PN10597 ) . The mean and standard deviation ( SD ) of the population medians of at least 200 , 000 cells in experimental triplicates are shown in black . The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes .

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Figure 1(B) Example images of brightfield and fluorescently labelled HPG (Alexa Fluor 647) of wild type cells (PN1) assayed for global cellular translation. Scale bars represent 5 µm. (C) Change in HPG labelling signal with different durations of HPG incubation (in PN1), measured by flow cytometry. Population medians of at least 200,000 cells are shown. The red line is the ordinary least square (OLS) linear regression fitted on the medians between 0 and 5 minutes. ((E) Example images of brightfield and fluorescently labelled EU (Alexa Fluor 488) of hENT1 and hsvTK cells (PN10567) assayed for global cellular transcription. Scale bars represent 5 µm. (F) Change in EU labelling signal with different lengths of EU incubations, measured by flow cytometry (in PN10597). The mean and standard deviation (SD) of the population medians of at least 200,000 cells in experimental triplicates are shown in black. The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes.


Figure 2 ( H ) Medians of global translation ( solid black line ) and IQR ( shaded area ) of cdc25 - 22 ( PN143 ) cells grown at 30 ° C and grouped in length bins of 2 µm . Bins containing more than 30 cells are shown . The solid red lines represent a locally estimated scatterplot smoothing ( LOESS ) function fitted on the single - cell data . ( I ) Global cellular translation of cells shown in ( H ) divided by their cell length . The solid black line represents the LOESS function fitted in ( H ) .

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Figure 2(H) Medians of global translation (solid black line) and IQR (shaded area) of cdc25-22 (PN143) cells grown at 30 °C and grouped in length bins of 2 µm. Bins containing more than 30 cells are shown. The solid red lines represent a locally estimated scatterplot smoothing (LOESS) function fitted on the single-cell data.(I) Global cellular translation of cells shown in (H) divided by their cell length. The solid black line represents the LOESS function fitted in (H).


Figure 3 ( D ) For each of the 5 length bins boxed in ( C ) , both 1C and 2C medians are normalised to their respective 1C global cellular translation values . The normalised values are represented as dots , each dot corresponding to one of the 5 length bins , the mean and SD of the normalised values are shown for each DNA content . For each DNA content , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( C ) . The p - value is calculated using a Welch ' s unequal variances t - test . ( F ) Example images of brightfield and EGFP - PCNA fluorescence in CCP ∆ EGFP - pcn1 cells ( PN6001 ) . The dashed lines in the EGFP - PCNA channel delimit the cell masks generated from the brightfield image . Cells with visible foci in the EGFP - PCNA channel are highlighted in yellow and marked with arrows . Scale bars represent 5 µm . ( K ) For each of the 4 length bins boxed in ( J ) , G1 , S , and G2 / M medians are normalised to their respective G1 global cellular translation value . The normalised values are represented as dots , each dot corresponding to one of the 4 length bins , the mean and SD of the normalised values are shown for each cell cycle stage . For each cell cycle stage , the normalised values ( dots ) are in the same order ( left to right ) as their corresponding length bins in ( J ) . The p - value is calculated using a Welch ' s unequal variances t - test .

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Figure 3(D) For each of the 5 length bins boxed in (C), both 1C and 2C medians are normalised to their respective 1C global cellular translation values. The normalised values are represented as dots, each dot corresponding to one of the 5 length bins, the mean and SD of the normalised values are shown for each DNA content. For each DNA content, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.(F) Example images of brightfield and EGFP-PCNA fluorescence in CCP∆ EGFP-pcn1 cells (PN6001). The dashed lines in the EGFP-PCNA channel delimit the cell masks generated from the brightfield image. Cells with visible foci in the EGFP-PCNA channel are highlighted in yellow and marked with arrows. Scale bars represent 5 µm.(K) For each of the 4 length bins boxed in (J), G1, S, and G2/M medians are normalised to their respective G1 global cellular translation value. The normalised values are represented as dots, each dot corresponding to one of the 4 length bins, the mean and SD of the normalised values are shown for each cell cycle stage. For each cell cycle stage, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (J). The p-value is calculated using a Welch's unequal variances t-test.