FIGURE 7.

5-aza affects ASC senescence. (A) Phase-contrast photomicrographs showing ASCs pretreated with 5-aza or DMSO at passage 4, 8 and 12 (P4-P12), stained with β-Galactosidase Staining Kit to visualize senescent cells. The percentage of β-Gal-positive cells was determined by counting the number of blue cells vs. total number of cells in ten random areas for each condition. (B) P53 and p21 mRNA expression, evaluated by qRT-PCR. mRNA levels were normalized to GAPDH mRNA expression. (C) Western blot analysis of p53, p21 and lamin B1 protein expression. β-Actin was used as internal control. Densitometric analysis was reported as relative expression with respect to DMSO-treated cells. (D) IF analysis showing the expression of Lamin B1 (green). Nuclei were visualized using DAPI (blue). Scale bar: 50 µm. Bars represent means ± SD of three independent experiments, each performed in triplicate. *p < 0.05, **p < 0.005 vs. DMSO.