Additional file 2 of Sustained CHK2 activity, but not ATM activity, is critical to maintain a G1 arrest after DNA damage in untransformed cells

Additional file 2: Fig. S2. CHK2 activity is required to maintain cell cycle arrest after DSB induction in G1, but not in G2. a RPE-1 cells irradiated with 4 Gy and treated with specific inhibitors for ATM and CHK2 show similar levels of total CHK2 protein. b Left panel: G1-synchronized RPE-1 cells were irradiated (4 Gy) and treated with the indicated doses of either CHK2i-II or CHK2i from Apex-Bio for one hour; protein extracts were analyzed by western blot. Right panel: G1 cells treated as in b with 10 µM and 0.5 µM concentrations of CHK2i-II or CHK2i-Apex-Bio, respectively, were further incubated with BrdU/STLC, and BrdU incorporation was analyzed by flow cytometry. c G2-synchronization protocol. Asynchronous RPE-1 cells were seeded, allowed to attach for approximately 24 hours, and blocked in the G1/S boundary with thymidine for 24 hours; cells were released for 7 hours to obtain a G2-enriched population. d PI profile of G2 cells shows that 90% of RPE-1 cells are in G2 phase after the synchronization protocol. e G2-synchronized RPE-1 cells were left unirradiated (0 Gy) or irradiated (4 Gy); irradiated cells were left untreated (UT) or treated with inhibitors for ATM (ATMi) or CHK2 (CHK2i) at the indicated times (0 or 1 hour after IR) and protein was harvested at 2 hours post-damage timepoint for western blot analysis. CDK4 served as a loading control. f G2-synchronized RPE-1 cells were treated with CHK2i or ATRi (positive control); BrdU and STLC were added at the time of IR, and cells were collected by trypsinization for flow cytometry analysis of mitotic cells that were in G2 at the time of IR (BrdU-negative/MPM2-positive). Statistical analysis was carried out using one-way ANOVA (n.s.: non-significant; **p