Figure 3: Activation of ATM-p53 pathway at the large foci of phosphorylated H2AX. (a) Investigation of factors relating to G1 checkpoint machinery in replicative senescence was performed by western blotting. “p-p53” indicated Ser15-phosphorylated p53. (b) p53 accumulation in cells formed phosphorylated ATM foci (p-ATM) in replicative senescence. (c) The cells detected different p53 level was scored at both PDL of 21 (closed bar) and of 64 (open bar) ( ). “−”, “+”, and “+++” indicated p53 negative, positive, and strong positive, respectively. Representative cells of p53 positive or strong positive were shown in upper or lower image in (b). (d and e) The cells were preextracted to remove chromatin-free nuclear protein, and then detected indicated proteins. (f) p53 phosphorylation at Ser15 was examined in senescent cells treated with a series of concentration of wortmannin.