Figure 6. ROS-triggered DNA damage response is correlated with PUMA-induced apoptosis.

(A) Inhibition effects of ATM/ATR/DNA-PK inhibitors were confirmed by western blotting. A2780s cells were untreated or were treated with 10 μM etoposide (to provide a positive control) in the presence or absence of ATM-specific inhibitor KU60019 (5 μM), ATR-specific inhibitor VE821 (1 μM) and DNA-PK-specific inhibitor NU7026 (10 μM) for 24 h, and then the levels of total and phosphorylated ATM, ATR, and DNA-PKcs were detected by western blotting. β-actin was used as a loading control. (B) Inhibition effects of caffeine were confirmed by western blotting. A2780s cells were untreated or were treated with 10 μM etoposide (to provide a positive control) in the presence or absence of PIKKs inhibitor caffeine (2 mM) for 24 h, and then the levels of total and phosphorylated ATM, ATR, and DNA-PKcs were detected by western blotting. β-actin was used as a loading control. (C) Effect of ATM/ATR/DNA-PK inhibitors on the phosphorylation of Chk1, Chk2 and H2AX. A2780s cells were untreated or infected with Ad-PUMA for 24 h in the presence or absence of KU60019 (5 μM), VE821 (1 μM), NU7026 (10 μM) and caffeine (2 mM) for an additional 12 h, and then the expressions of total and phosphorylated Chk1 and Chk2 and H2AX were detected by western blotting. β-actin was used as a loading control. (D) A2780s cells were treated as described in B. Then the treated cells were used for analysis of apoptosis by flow cytometry. (E) The percentage of apoptotic cells was quantified. Bars, mean; error bars, S.D. (n = 3; **p < 0.01; N.S., not significant). (F) A2780s cells were treated as described in B. The treated cells were then lysed and caspase 3 activity was measured using an assay kit. Bars, mean; error bars, S.D. (n = 3; **p < 0.01; n.s., not significant).