Fig. 1

Double-strand break (DSB) induction in G1 by irradiation activates ATM/CHK2/p53-dependent signaling and triggers cell cycle arrest. a RPE-1 cells were fixed at the indicated times after 4 Gy IR. EdU was added at the time of IR and cells were stained for EdU using Click-iT chemistry after IF for DAPI, γH2AX, and 53BP1. The number of γH2AX and 53BP1 foci per nucleus was counted with an in-house developed macro in G1 cells, which were identified by the nuclear size (small DAPI intensity) and absence of EdU staining. b G1-synchronized RPE-1 cells were left untreated (0 Gy) or treated with two IR doses (2 Gy, 4 Gy), or four NCS doses (10, 20, 40, 80 ng/ml); protein was collected 2 or 16 h after DNA damage for the detection of the indicated proteins by western blot. Phosphorylation of CHK2 on T68 and S516 are indicative of active ATM and CHK2 kinases; increased p53 proteins levels are indicative of stabilized and active p53; CDK4 served as a loading control. c G1-synchronized RPE-1 cells were treated as in b; BrdU and STLC were added at the time of IR/NCS, and cells were collected by trypsinization 40 h later for flow cytometry analysis (upper panel). The percentage of BrdU-positive cells is indicative of the percentage of cells that have entered into S-phase, and decreases with IR in a dose-dependent manner. Depicted are the means and standard deviation of three independent experiments (lower panel)