F Plasma levels of cardiac stress biomarkers : brain natriuretic peptide ( BNP ) ( F1 ) and cardiac troponin I ( cTnI ) ( F2 ) following isoproterenol treatment in mdx NaCl ( n = 5 ) , surviving mdx ( n = 8 ) , mdx / CD38 - / - NaCl ( n = 5 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : in duplicate After normality and variance comparison tests , significance was assessed using F2 Kruskal - Wallis followed by Mann - Whitney tests ; F1 : ANOVA followed by Student ' s / Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

F Plasma levels of cardiac stress biomarkers: brain natriuretic peptide (BNP) (F1) and cardiac troponin I (cTnI) (F2) following isoproterenol treatment in mdx NaCl (n=5), surviving mdx (n=8), mdx/CD38-/- NaCl (n=5) and mdx/CD38-/- (n=8) mice. Data information: in duplicate After normality and variance comparison tests, significance was assessed using F2 Kruskal-Wallis followed by Mann-Whitney tests; F1: ANOVA followed by Student's/ Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


G , H Time - lapse images recorded by confocal microscopy in " line scanning mode " , showing Ca2 + sparks and waves in cardiomyocytes at rest , extracted from mdx and mdx / CD38 - / - mice ; scale bars : 10 µM ( horizontal ) , 500 ms ( vertical ) . Bar graphs showing the averaged Ca2 + sparks ( G ) and waves ( H ) frequencies in cardiomyocytes isolated from WT ( n = 21 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using Kruskal - Wallis followed by Dunn ' s test ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

G,H Time-lapse images recorded by confocal microscopy in "line scanning mode", showing Ca2+ sparks and waves in cardiomyocytes at rest, extracted from mdx and mdx/CD38-/- mice; scale bars: 10 µM (horizontal), 500 ms (vertical). Bar graphs showing the averaged Ca2+ sparks (G) and waves (H) frequencies in cardiomyocytes isolated from WT (n=21 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 33 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : ANOVA followed by Fisher ' s LSD test ; ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT (n=24 cells), mdx (n=33 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


J Bar graph showing the post - rest potentiation in cardiomyocytes from WT ( n = 24 cells ) , mdx ( n = 28 cells ) and mdx / CD38 - / - ( n = 32 cells ) mice . Data information : experiments performed in 3 mice of each group . After normality and variance comparison tests , significance was assessed using : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

J Bar graph showing the post-rest potentiation in cardiomyocytes from WT (n=24 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


A Measurement of the ventilatory mechanic by barometric plethysmography : dot plots showing inspiratory ( A1 ) and expiratory times ( A2 ) , the relaxation time ( A3 ) and the respiratory frequency ( A4 ) in WT ( n = 8 ) , mdx ( n = 11 ) and mdx / CD38 - / - ( n = 9 ) mice . Data information : Each dot of the graphs represents a mouse . one value / mouse After normality and variance comparison tests , significance was assessed using : A1 , A3 ANOVA followed by Fisher ' s LSD test ; A2 , A4 : Kruskal - Wallis followed by Dunn ' s test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A Measurement of the ventilatory mechanic by barometric plethysmography: dot plots showing inspiratory (A1) and expiratory times (A2), the relaxation time (A3) and the respiratory frequency (A4) in WT (n=8), mdx (n=11) and mdx/CD38-/- (n=9) mice. Data information: Each dot of the graphs represents a mouse. one value/mouse After normality and variance comparison tests, significance was assessed using: A1,A3 ANOVA followed by Fisher's LSD test; A2, A4 : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


B Images showing muscle fiber typology revealed by immunostaining . Localization of the slow MyHC ( Type I ) fiber and fast MyHCs ( Type IIa and IIb / X ) fibers , along with laminin ( red ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 100 µm . Histogram showing the percentage of I , IIa and IIb / X fiber type distribution in diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : ; B : Chi - square test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images showing muscle fiber typology revealed by immunostaining. Localization of the slow MyHC (Type I) fiber and fast MyHCs (Type IIa and IIb/X) fibers, along with laminin (red) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 100 µm. Histogram showing the percentage of I, IIa and IIb/X fiber type distribution in diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: ; B: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


C Fiber size distribution in the diaphragm of WT ( n = 7 ) , mdx ( n = 6 ) and mdx / CD38 - / - ( n = 7 ) mice . Data information : one value / mouse After normality and variance comparison tests , significance was assessed using : C : Kolmogorov - Smirnov test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C Fiber size distribution in the diaphragm of WT (n=7), mdx (n=6) and mdx/CD38-/- (n=7) mice. Data information: one value/mouse After normality and variance comparison tests, significance was assessed using: C: Kolmogorov-Smirnov test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


D Images revealing the collagen ( blue ) by Masson ' s trichrome staining in diaphragm of WT , mdx , and mdx / CD38 - / - mice . Quantification of collagen staining area ( % total area ) in the diaphragm of WT , mdx and mdx / CD38 - / - mice ( n = 4 per group ) . Scale bars : 200 µm . Data information : Each dot of the graphs represents a mouse . D in duplicate ; After normality and variance comparison tests , significance was assessed using : : ANOVA followed by Fisher ' s LSD test Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

D Images revealing the collagen (blue) by Masson's trichrome staining in diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


E Embryonic myosin expression : immunostaining showing its localization along with laminin ( green ) on transverse cross - sections from diaphragm of WT , mdx and mdx / CD38 - / - mice . Scale bars : 50 µm . Histogram showing the relative proportion of embryonic myosin area ( % total area ) in diaphragm of WT ( n = 7 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 8 ) mice . Data information : Each dot of the graphs represents a mouse . E one value / mouse . After normality and variance comparison tests , significance was assessed using : E : Welch ' s ANOVA followed by Welch ' s t - tests . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E Embryonic myosin expression: immunostaining showing its localization along with laminin (green) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 50 µm. Histogram showing the relative proportion of embryonic myosin area (% total area) in diaphragm of WT (n=7), mdx (n=8) and mdx/CD38-/- (n=8) mice. Data information: Each dot of the graphs represents a mouse. E one value/mouse. After normality and variance comparison tests, significance was assessed using: E: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


A Images showing immunostaining of myeloid cells showing F4 / 80 ( macrophage marker ) , Ly - 6G / 6C ( monocyte , granulocyte and neutrophil marker ) , CD8 ( cytotoxic T lymphocyte marker ) and IL - 6 positive cells in diaphragm of 7 - month - old WT ( n = 8 ) , mdx ( n = 8 ) and mdx / CD38 - / - ( n = 7 or 8 ) mice . Lower panel : histograms quantifying the percentage of F4 / 80 ( A1 ) mdx / CD38 - / - ( n = 7 ) , LY6 ( A2 ) mdx / CD38 - / - ( n = 8 ) , CD8 ( A3 ) mdx / CD38 - / - ( n = 7 ) , IL - 6 ( A4 ) mdx / CD38 - / - ( n = 8 ) positive cells . Scale bars : 50 µm . Data information : Each dot of the graphs represents a mouse . A one value / mouse After normality and variance comparison tests , significance was assessed using : A1 - 4 : Welch ' s ANOVA followed by Welch ' s t - tests ; Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A Images showing immunostaining of myeloid cells showing F4/80 (macrophage marker), Ly-6G/6C (monocyte, granulocyte and neutrophil marker), CD8 (cytotoxic T lymphocyte marker) and IL-6 positive cells in diaphragm of 7-month-old WT (n=8), mdx (n=8) and mdx/CD38-/- (n=7 or 8 ) mice. Lower panel: histograms quantifying the percentage of F4/80 (A1) mdx/CD38-/- (n=7), LY6 (A2) mdx/CD38-/- (n=8), CD8 (A3) mdx/CD38-/- (n=7), IL-6 (A4) mdx/CD38-/- (n=8) positive cells. Scale bars: 50 µm. Data information: Each dot of the graphs represents a mouse. A one value/mouse After normality and variance comparison tests, significance was assessed using: A1-4: Welch's ANOVA followed by Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


B qPCR analysis of mRNA levels of interleukin - 1β ( IL - 1ß ) ( B1 ) and - 6 ( IL - 6 ) ( B2 ) , cyclin - dependent kinase inhibitor 1 ( p21 ) ( B3 ) transforming growth factor - β ( TGF - β ) ( B4 ) , and senescence markers ( cell - cycle inhibitor p16 , INK4a ) ( B5 ) , Collagen Type I Alpha 1 Chain ( Col1a1 ) ( B6 ) in diaphragm of 20 - month - old WT ( n = 6 ) , mdx ( n = 5 ) and mdx / CD38 - / - ( n = 5 ) mice . Data information : Each dot of the graphs represents a mouse . B in duplicate . After normality and variance comparison tests , significance was assessed using : B1 - 6 : ANOVA followed by Fisher ' s LSD test . Values are expressed as means ± SEM . Significance : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR analysis of mRNA levels of interleukin-1β (IL-1ß) (B1) and -6 (IL-6) (B2), cyclin-dependent kinase inhibitor 1 (p21) (B3) transforming growth factor-β (TGF-β) (B4), and senescence markers (cell-cycle inhibitor p16, INK4a) (B5), Collagen Type I Alpha 1 Chain (Col1a1) (B6) in diaphragm of 20-month-old WT (n=6), mdx (n=5) and mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse. B in duplicate. After normality and variance comparison tests, significance was assessed using: B1-6: ANOVA followed by Fisher's LSD test. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.


A 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 .

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

A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

C 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 .

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

D 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 .

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

E 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 .

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

F 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 .

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.


A 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 .

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

A 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 - / - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 - / - .

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

C 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 .

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

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 .

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

E, 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 .

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

F 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 .

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

G 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 .

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

H 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


( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 in H1299 cells in normal medium ( HBSS ' ' - ' ' ) or after 1 hr HBSS starvation ( HBSS ' ' + ' ' ) pretreated with or without NAC ( 3mM ) .

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

(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).


( B ) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment .

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

(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.


( C ) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 ( 500μM ) treatment . CHK2 NTm , a shRNA nontargetable mutant CHK2 rescue plasmid .

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

(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.


( D ) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably - expressing GFP - mCherry - LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 ( 500μM ) treatment for 3h . Scale bar , 10 μm .

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

(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.


( E ) Quantitation of autophagosomal ( yellow ) and autolysosomal ( red ) LC3 puncta following HBSS starvation and H2O2 ( 500μM ) treatment for 1h ( n = 30 ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 ( Student ' s t - test ) .

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

(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).


( F ) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Scale bars , 500 nm . The red arrows indicate double - membraned autophagic structures .

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

(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.


( G ) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ; * * P < 0 . 01 ( Student ' s t - test ) .

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

(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).


( A ) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy - related genes in HCT116 cells . Lysates were extracted for immunoprecipitation with CHK2 - specific antibody or control IgG , followed by probing with antibodies specific for Ulk1 , Atg5 , Beclin1 , Atg7 or LC3 .

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

(A) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy-related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2-specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin1, Atg7 or LC3.


( B ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after HBSS starvation in the cytoplasmic fractions .

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(B) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after HBSS starvation in the cytoplasmic fractions.


( C ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after H2O2 ( 500μM ) stimulation in the cytoplasmic fractions .

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

(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.


( D ) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

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

(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation ( E ) The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

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

HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 ( 500μM ) stimulation ( F ) . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

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

HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were untreated ( G ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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

HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were untreated (G) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with HBSS starvation ( H ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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

HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with HBSS starvation (H) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with H2O2 ( 500μM ) stimulation ( I ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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

HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


( J ) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 ( 500 μM ) cultured for 3 hr .

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

(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.


( K ) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 ( 500μM ) stimulation . Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 .

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

(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.


( A ) The indicated recombinant fragments of GST - Beclin1 were incubated with recombinant CHK2 in the presence of 32P - labeled ATP for in vitro kinase assays . MBP was used as a positive control . Ponceau staining indicates the expression of GST - Beclin 1 fragments for in vitro kinase assays . The asterisks in the blot indicate the domain of phosphorylated Beclin 1 .

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

(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.


( C ) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST - Beclin 1 - WT , S9093A ( AA ) , S90A and S93A protein . Reactions were analyzed by SDS - PAGE followed by autoradiography . The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants .

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

(C) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST-Beclin 1-WT, S9093A (AA), S90A and S93A protein. Reactions were analyzed by SDS-PAGE followed by autoradiography. The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants.


Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation ( D )

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

Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D)


Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 ( 500μM ) stimulation ( E ) .

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

Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).


Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation ( F )

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

Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F)


Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 ( 500μM ) stimulation ( G ) .

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

Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 (500μM) stimulation (G).


( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation .

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

(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.


( A ) The effect of Beclin 1 Ser90 / 93 phosphorylation on the interaction between Beclin 1 and Bcl - 2 . GST - Beclin1 WT , AA mutant and DD mutant ( bait ) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag - Bcl - 2 ( preys ) .

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

(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).


( B ) Immunoprecipitation of Bcl - 2 with Flag - Beclin 1 WT , AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 ( 500μM ) treatment for 1h .

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

(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.


( C - D ) Autophagy levels were determined in Beclin 1 - depleted H1299 cells with reconstituted expression of Beclin 1 WT , AA mutant or DD mutant in normal medium or after H2O2 ( 500μM ) treatment for 3h in the presence ( C ) or absence ( D ) of CHK2 .

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

(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.


( E ) Representative electron microscopic image of an autophagosome ( arrow , left panel , AP ) and an autolysosome ( arrow , right panel , AL ) in H1299 Beclin 1 WT cells treated with H2O2 ( 500μM ) for 6 hr . Scale bars , 500 nm . Electron microscopic quantification of autophagosomes ( AP ) and autolysosomes ( AL ) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 ( 500μM ) treatment . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 001 compared to Beclin 1 WT ; # P < 0 . 001 compared to Beclin 1 WT treated with H2O2 ; & P < 0 . 001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2 .

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

(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.


( F ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 , Beclin 1 , p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment for 3h .

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

(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.


( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation .

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

(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.


( B ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia .

O O O O O O O O O O O O O O O O O O O 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) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.


( C ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation .

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

(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.


( D ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia .

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

(D) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.


( E ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to hypoxia with or without pretreatment with NAC .

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

(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.


( F ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to glucose starvation with or without pretreatment with NAC .

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

(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.


( G ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , and AMPKα in H1299 cells in normal medium or after H2O2 ( 25μM ) stimulation .

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

(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.


( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG ( 5mM ) and oligomycin ( 2 . 5μM ) treatment .

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

(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.


Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation ( Glu - )

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

Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-)


Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia ( B ) .

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

Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B).


( C ) Western blot detection of CHK2 , HSP60 , TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation ( Glu - ) or 36 hr hypoxia .

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

(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.


( D - E ) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation ( Glu - ) medium or hypoxia . The yellow asterisks mark mitochondria . The red arrows indicate autophagic structures . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 compared to shNC treated with H2O2 or hypoxia ( Mann - Whitney test ) ; Scale bar , 1 μm .

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

(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.


( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm . Scale bar , 10 μm .

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

(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm.


The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation ( G ) All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia ( H ) . All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation ( A ) Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments ; * p < 0 . 05 compared to NC glucose starvation ( A ) ( Student ' s t - test ) .

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

H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).


H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation ( B ) . Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments * * p < 0 . 01 compared to NC hypoxia ( B ) ( Student ' s t - test ) .

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

H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments **p < 0.01 compared to NC hypoxia (B) (Student's t-test).


H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation ( C ) Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) .

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

H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation ( D ) . Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) .

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

H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


( E ) CHK2 + / + and CHK2 - / - mice were subjected to MCAO for 1 hr and reperfusion for 12 h . Contralateral ( C ) and ipsilateral ( I ) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC .

(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.


( F ) The infarct volume was determined by measuring infarct size relative to normal in the slice . ( n = 5 mice ) . Data are presented as mean ± s . e . m . ; * * P < 0 . 01 ( Student ' s t - test ) .

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(F) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).


( G - J ) Immunoblots for p62 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion - treated CHK2 + / + and CHK2 - / - mice . Quantification of p62 , p - Beclin 1 Ser90 and p - Beclin 1 Ser93 protein levels ( n = 3 mice ) . Data are presented as mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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(G-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).


( A ) NPM1 expression in CD34 + Lin - cells from myelodysplastic syndrome ( MDS ) patients compared to healthy volunteers ( n = 29 ) .

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(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29).


( B ) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia ( AML ) with multi - lineage dysplasia ( MLD ) . Insets show representative staining in blasts [ B ] and in differentiated myeloid cells [ e . g . , neutrophil , M ] with a high magnification .

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(B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification.


( C ) Percentages of patient samples with NPM1 - low and NPM1 - normal in healthy , MDS and MLD in AML groups .

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(C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups.


( C ) Representative distribution of Tubulin and CDC42 in Ctrl , 4 - months old Npm1 cKO and 2 - years old WT HSCs determined by IF . Scale bars , 5 μm .

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(C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm.


( F ) HSCs of Ctrl , 4 - months old Npm1 cKO and 2 - years old WT mice were sorted and stained for TOM20 . Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) in single cells ( n = 58 cells in Ctrl , n = 70 cells in cKO and n = 23 cells in Old , from 3 mice per group ) , identified by volume rendering of TOM20 . Scale bars , 2 μm .

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(F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm.


( J ) Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) , identified by volume rendering of TOM20 , in MDS - L cells transfected with siNPM1 or control ( siCTRL ) ( n = 58 cells per group , from 3 transfected MDS - L cultures ) . Scale bars , 2 μm .

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(J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm.


( F ) Representative peripheral blood ( PB ) smears . Insets show the differentiated granulocytes ( a , b ) in control mice , and anisocytosis ( c ) , dysplastic myeloid cells ( d - g ) in Npm1 cKO mice ( Npm1F / FVav1Cre + ) . Npm1 cKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ; schistocyte , blue - outlined arrowhead ; tear drop , Howell - Jolly bodies , black arrowhead ; black - outlined arrowhead ) , dysplastic monocytes ( black arrow ) , and dysplastic neutrophils ( d - f ) . Scale bars , 10 μm .

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(F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm.


( C ) Npm1 and p53 protein levels in KSL cells of Ctrl ( Npm1F / FVav1Cre - ) , and cKO ( Npm1F / FVav1Cre + ) mice .

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(C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice.


( F ) Representative PB smears of DKO mice . Insets show anisocytosis ( a ) , poikilocytosis ( b ) and dysplastic myeloid cell ( c ) . DKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ) , Howell - Jolly bodies , ( black arrowhead in insert a ) , dysplastic monocytes ( black arrow , and i - v ) , dysplastic neutrophils ( vi ) , a pseudo - Pelger - Huet anomaly ( ii ) , and dysplastic platelets ( giant platelet , iv and vii ) . Scale bars , 10μm .

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(F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm.


( I ) Representative H & E staining of sections of the spleen of 2 - month - old DKO mice with myeloid disorders . Arrowheads ( right ) indicate the infiltrating myeloid blasts . Scale bars , 250 μm ( left ) and 10μm ( right ) , respectively .

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(I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively.


B . Growth comparisons of selected kinases with the kinase ‑ dead mutant match . 24h growth curves ( OD595 ) from liquid culture of yeast strains ( W303 ) expressing human kinase in the presence or absence of 3 . 2 % ( v / v ) DMSO . Upper panel : BUB1 : BUB1 wild type , BUB1 - KD : BUB1 ( K821M ) ; upper middle panel : NEK6 : NEK6 wild type , NEK6 - KD : NEK6 ( K74M & K75M ) ; lower middle panel : PKCα - ac : PKCα ( A25E ) , PKCα - KD : PKCα ( K368R ) ; lower panel : Control with empty plasmid . Error bars represent SD from three biological replicates .

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B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.


C . Human protein kinases phosphorylate large sets of yeast proteins . Upper panel : In total , 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho - motif antibodies on yeast proteins in comparison to other human kinases , kinase ‑ dead mutant versions or vector control samples . Commercially available pS / T - antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases . Lower panel : Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded . When developed with the indicated phospho - motif recognizing antibodies , immunoreactive bands indicated phosphorylation of yeast proteins .

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C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.


C . Y2H interaction experiment with human AAR2 ( FL ) as bait and PRP8 ( aa1755 - 2335 ) as prey . Growth on selective media is abolished when AAR2 carries a S284E phospho - mimicry amino acid substitution . Mutation of second candidate phospho - site , AAR2 ( T356 ) , does not influence the AAR2 - PRP8 protein interaction .

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C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.


D . Interaction of AAR2 and PRP8 in human cells . HA ‑ tagged PRP8 ( aa1755 - 2335 ) was co - expressed in HEK293 cells with either wildtype AAR2 , AAR2 ( S284A ) or AAR2 ( S284E ) mutant versions . Immunoprecipitation of PRP8 led to a co - precipitation of wild typeAAR2 and phospho - null AAR2 ( S284A ) . In contrast , the phospho - mimicry AAR2 ( S284E ) version did not bind PRP8 .

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D. Interaction of AAR2 and PRP8 in human cells. HA‑tagged PRP8 (aa1755-2335) was co-expressed in HEK293 cells with either wildtype AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a co-precipitation of wild typeAAR2 and phospho-null AAR2(S284A). In contrast, the phospho-mimicry AAR2(S284E) version did not bind PRP8.


E . Results of the phospho - Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8 . Growth on selective media ( SD5 ) , of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co - expression of either SGK2 or CK2α1 ( CSNK2A1 ) . Growth reduction is visible in a 1 : 10 dilution series on selective agar ( SD5 ) in comparison to the control media ( SD3 ) . Ctrl : non - related kinases .

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E. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.


F . Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases . Experiment as in D . Co - expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co - immunoprecipitated with RPR8 .

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F. Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases. Experiment as in D. Co-expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co-immunoprecipitated with RPR8.


B . Results of the phospho - Y2H screen to identify kinases modulating the interaction of the ERα with 14 ‑ 3 ‑ 3β . The interaction of ERα ( prey ) with 14 ‑ 3 ‑ 3β ( bait ) is promoted by the co - expression of wildtype kinases of PAK5 , CDK17 , BUB1 and NEK6 , visible by the yeast growth on selective media in comparison to the control ( three biological replicas are shown ) . The coexpression of the kinase ‑ dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14 ‑ 3 ‑ 3β . In contrast the kinase ‑ dead mutant versions of BUB1 ( K821M ) and NEK6 ( K74M & K75M ) did not promote the Y2H interaction .

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B. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction.


C . Phosphorylation of ERα in mammalian cells . ERα was co - expressed with the indicated wildtype kinases or kinase ‑ dead versions in HEK293 cells and immunoprecipitated . Phosphorylation status of ERα was analyzed with an in - gel phosphostain ( Pro - Q ® Diamond phosphoprotein gel stain , Invitrogen ) , relative to the whole protein stain ( SYPRO ® Ruby protein gel stain , Invitrogen ) .

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C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).


D . Interaction of ERα and 14 ‑ 3 ‑ 3β in a GST pull - down assay . GST ‑ tagged 14 ‑ 3 ‑ 3β was expressed in E . coli and immobilized on beads . PA ‑ tagged ERα was co - expressed with either wildtype PAK5 or NEK6 , the kinase ‑ dead ( KD ) version or without a kinase in HEK293 cells . Immobilized 14 ‑ 3 ‑ 3β was used to pull PA ‑ tagged ERα from the HEK lysates . Co - precipitation of ERα was successful in the presence of wild kinases , but not in their absence or with kinase ‑ dead mutant versions .

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D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions.


E . Effect of NEK6 co - expression on ERα gene activation . Relative luciferase activity of ERα in the presence and absence of 1 nM E2 ( 17β - estradiol ) was measured . A dose ‑ dependent reduction ( ng plasmid transfected ) in E2 - Erα ‑ dependent luciferase expression was observed in HEK293T , MCF - 7 and US - OS cancer cell lines . This effect was not observed with the NEK6 ( K74M & K75M ) kinase ‑ dead version . Graphs represent ≥ 5 biological replicates , each derived from averaging 6 technical replicates . Error bars indicate SD across the biological replicates .

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E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.


( A ) Unlabeled or Cy5 - labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase ( dsRNA - RL ) or the GFP ( dsRNA - GFP ) coding sequences visualised on gel stained with ethidium bromide ( EtBr - stained , top ) or by in - gel fluorescence ( bottom ) .

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(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).


( B ) Gating strategy used in the flow cytometry - based assay . First , cells were selected ( side scatter ( SSC ) vs . forward scatter ( FSC ) ) and gated for singlets ( SSC - Width ( W ) vs . FSC ) to hence exclude doublets or cell aggregates . The single cell gate was then further analysed for DAPI , gating on the live cells ( DAPI negative ) . DsRNA - transfected cells ( Cy5 - positive ) were then gated and the level of GFP expression analysed within that population . Flow cytometry counting beads ( gated on SSC vs . FSC plot ) were added to quantify cell number in each sample .

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(B) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample.


( C ) mESCs stably expressing GFP ( mESCs - GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and the level of GFP in Cy5 + cells was analysed 3 days later and is represented as histograms ( left ) . Right panel , bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control ( D ) mESCs stably expressing d2GFP ( mESCs - d2GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA . The level of d2GFP was analysed at the indicated timepoints post - transfection and displayed as in ( C ) .

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(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).


( E ) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and d2GFP level was measured as in ( D ) .

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(E) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).


( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs ( top panels ) or siRNAs ( bottom panels ) . GFP level was measured in Cy5 + cells by flow cytometry 72 hours ( hrs ) post - transfection .

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(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.


( B ) Mavs + / - and Mavs - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs . The level of d2GFP in Cy5 + cells was measured at various timepoints post - transfection as indicated . Histograms plots show d2GFP level 48 hrs post - transfection . Bar graphs show 3 timepoints as indicated .

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(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.


( C ) WT , Ifnar1 + / - or Ifnar1 - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry at 48 hrs post - transfection .

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(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.