Figure 3. Oxidative stress-induced cellular senescence is reduced by genetic depletion of Atm. (A) Proliferation of WT (black), Ercc1-/- (blue) and Ercc1-/-Atm+/- (red) MEFs serially passaged at 20% oxygen was measured by an automated cell counter. Data shown are representative of three independent experiments using distinct MEF lines. (B) SA-βgal staining of serially passaged MEFs cultured at 20% oxygen. Shown are representative images of passage 5 WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs taken at 10x magnification. (C) The average percentage of SA-βgal positive cells at each indicated passage. Ten fields were acquired and quantified per sample. Data shown are representative of two independent experiments. (D) Senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs (passage 5) cultured at 20% oxygen for 72 hrs were collected and lysed for immunoblot analysis of p16INK4a. (E) Supernatant collected from senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs was analyzed by ELISA for secreted IL-6. Graphs represent mean+/- s.e.m. P value was determined using Student’s t-test. *p<0.05, **p<0.01.