Fig. 3.

mTORC1 regulates stress-induced senescence via p70S6K. a β-gal staining of control siRNA- or p70S6K-siRNA-treated hUC-MSCs in the presence or absence of Dox. 1 × 10⁵ hUC-MSCs (p5-p7) were seeded in 6-well plates and transfected with siRNAs. Twenty-four hours later, the cells were treated with Dox and collected 2 days afterward. Quantification data is shown below. Data are presented as the mean ± SEM. **p < 0.01;***p < 0.001 by one-way ANOVA with Tukey’s post hoc test. b Representative Western blot showing that knockdown of p70S6K alleviates Dox-induced DNA damage response. Cells were treated with Dox and collected 24 h later. Data are presented as the mean ± SEM. **p < 0.01;***p < 0.001 by one-way ANOVA with Tukey’s post hoc test. c The β-Gal staining shows that MHY1485 aggravates Dox-induced cellular senescence (scale bar = 100 μm). 2 × 10⁵ hUC-MSCs (p8-p9) were seeded in 6-well plates and treated with Dox with or without MHY1485. The cells were collected 2 days afterward. The quantification of β-Gal staining is shown below. Data are presented as the mean ± SEM. **p < 0.01;***p < 0.001 by one-way ANOVA with Tukey’s post hoc test. d 1 × 10⁴ hUC-MSCs (p8-p9) were seeded on coverslip and treated with Dox with or without MHY1485. The cells were collected 24 h afterward. Representative images and quantification of immunofluorescence staining of γ-H₂AX in control or MHY1485-treated hUC-MSCs in the presence or absence of Dox (scale bar = 10 μm). Quantification is shown below, mean ± SEM of values from three independent experiments with triplicate wells analyzed on 6–8 cells/field from five different fields. **p < 0.01 one-way ANOVA with Tukey’s post hoc test