Figure 3. The LY treatments cause modulation of both senescent phenotype and the phosphorylation status of mTOR targets and ERK1/2 MAPK in senescent MESCs. (A) SA-β-Gal staining 7 days after 1h-H2O2-treatment (middle) or after (H2O2+LY) treatment as described in the legend of Figure 2 (right). Ctr – control cells. Senescent cells were detected with SA-β-Gal staining kit. Ob: 10x; scale bars: 140 μm. (B) Quantitative assay of SA-β-Gal positive cells. Data are presented as mean ± SD, p < 0.001. (C, D) The expression levels of mTORC1 targets (p70S6K, S6, 4EBP-1), Akt, and ERK1/2 were revealed by immunoblot analysis 7 days after H2O2 or (H2O2+LY) treatment as indicated in (A). GAPDH was used as a loading control. Ctr – untreated cells. Representative results of the three experiments are shown in the figures. (E) Forward scatter (FS), reflecting the average cell size was measured by light-scattering cytometry 5 days after H2O2 or (H2O2+LY) treatment as indicated in (A). The results are presented as mean ± SD of three independent experiments, p<0.01 compared to control () and H2O2-treated cells (§). Abbreviations: pp85S6K, phospho-p85 S6 kinase (Thr412); pp70S6K, phospho-p70 S6 kinase (Thr389); pS6, phospho-S6 ribosomal protein (Ser240/244); p4EBP-1, phospho-4EBP-1 (Thr37/46); pAkt, phospho-Akt (Thr308); pERK1/2, phospho-ERK1/2 (Thr202/Tyr204).