Fig. 6: STS accelerates replicative senescence in primary human VSMC.
a Cell proliferation (BrdU incorporation) in vascular smooth muscle cells (VSMC) treated or not (Ctrl; 0 mM) with sodium thiosulfate (STS), as indicated. P values as determined by repeated measures (matched) mixed-effects model (REML) with followed by Dunnett’s multiple comparisons tests in 3 independent experiments. Scale bars are 50 μm. b, c Representative Western blot analysis of Cyclin B1, γH2AX, p21CIP, P-AMPK, AMPK, and IL-1β in VSMC treated for 24 h (b) or 48 h (c) with increasing concentration of STS, as indicated. Representative blots (left panels) and quantitative assessment (right panels) of protein expression, relative to total protein content, in 4 to 5 independent experiments. P values as determined by repeated measures mixed-effects model (REML) with followed by Dunnett’s multiple comparisons tests. dUpper panels: Representative P21CIP immunocytochemistry (green) and nuclei (DAPI; blue) in VSMC treated for 24 h with 10 mM STS. Lower panels: quantitative assessment of P21+ cells and nuclei size. Data are pooled scatter plots with mean ± SEM of 4 independent experiments. P values as determined by one-way ANOVA followed by Dunnett’s multiple comparisons tests. e Mitochondrial stress test assay (Seahorse) in VSMC pre-treated or not (Ctrl) for 4 h with 100 µM NaHS or 10 mM STS. Representative traces (left panel) and quantitative assessment (right panels) in 6 independent experiments. P-values as determined by repeated measures mixed-effects model (REML) with followed by Dunnett’s multiple comparisons tests. f Representative images and quantitative assessment of live Mitotracker staining in VSMC treated for 24 h with 10 mM STS. Data are pooled scatter plots of five independent experiments. P-values as determined by Kolmogorov-Smirnov test.
