Fig. 8
Cellular Model of Oxidative Stress-Induced Senescence in SK-MES-1 Cells. The cells were treated for 2 h without (0µM) or with 25µM and 50µM H2O2, and then treated with supplemented medium for an additional 72 h. A Cytochemical analysis of SA-β-gal activity; the lung SCC cells were stained for SA-β-gal and photographed with a phase contrast microscope at 200× magnification. B Flow Cytometry Gating for Single and Live Cells, Quarter 1: Forward Scatter Area vs. Forward Scatter Height; first gate that was used to exclude any doublets and clumps of multiple cells to eliminate skewed results, Quarter 2: Forward Scatter Area vs. Propidium iodide; gate that is used for live cells and gate out obvious debris (as dead cells often have varied areas and heights that can make data look sloppy), once the single cells of interest were the only cells in our analysis, Quarter 3: Control cells stained with propidium iodide show G1, S, and G2/M phase distributions. Quarter 4: The frequency of G1 senescent cells increased dramatically, and the frequencies of S and G2/M phases decreased significantly. Plots were made using ModFit. C Non-significant decreased ZNF334 expression in senescent cells treated with H2O2. qRT-PCR analysis was performed using RNA prepared from senescent SK-MES-1 cells treated with 25µM and 50µM H2O2 concentrations. GAPDH was used as a reference gene. Data are expressed using the 2−∆∆Ct method. Non-parametric Wilcoxon test was carried out to evaluate statistical differences between groups. Differences were considered significant when p < 0.05, D Significant increased TINAGL1 expression (p value25µM 0.0313, p value50µM 0.0313) in senescent cells treated with H2O2. qRT-PCR analysis was performed using RNA prepared from senescent SK-MES-1 cells treated with 25µM and 50µM H2O2 concentrations. GAPDH was used as a reference gene. Data are expressed using the 2−∆∆Ct method. Non-parametric Wilcoxon test was carried out to evaluate statistical differences between groups. Differences were considered significant at p < 0.05