Figure 3.

Impact of p53 and ATM on DSB-induced autophagy. (a) Time course of DNA damage response signaling and autophagy induction in isogenic p53-proficient and -deficient HCT116 cells. HCT116-p53^+/+ and HCT116-p53^−/− cells were challenged with CDT for up to 72 h. Cell lysates were then subjected to SDS-PAGE followed by western blotting detection as indicated. Hsp90 served as loading control. (b) Determination of autophagic vesicles in HCT116-p53^+/+ and HCT116-p53^−/− cells following DSB induction. Cells were irradiated or exposed to CDT as indicated and incubated for 24 h. Autophagosomes were stained with CytoID reagent and monitored by flow cytometry. (n=3); NS: not significant. *P<0.05, **P<0.01. (c) siRNA-mediated knockdown of p53 and accumulation of LC3B. HCT116 cells were transiently transfected with scrambled (scr) or p53-specific siRNA. Twenty-four hours after transfection, cells were treated with 500 ng/ml CDT or exposed to IR (10 Gy) and incubated for 48 h. Whole-cell extracts were analyzed by immunoblot detection using antibodies against p62 and LC3B. Hsp90 served as loading control, whereas p53 was visualized to confirm efficient knockdown. (d) Autophagosome formation upon siRNA-mediated depletion of p53. HCT116-p53^+/+ cells were transfected with p53 siRNA or scrRNA and treated as described above. Autophagic vesicles were detected by CytoID staining and flow cytometry. (n=3); NS: not significant. *P<0.05. (e) Effect of ATM inhibition on DSB-induced autophagosome formation. Cells were pretreated with the pharmacological ATM inhibitor KU-55933 (ATMi) and then exposed to either CDT or IR. After 48 h, autophagosomes were stained as described before (n=3); NS: not significant. *P<0.05. (f) Effect of ATM on DSB-dependent LC3B accumulation. Cells were treated as mentioned above and subjected to SDS-PAGE followed by western blotting analysis