Fig. 6

Loss of CHK2 activity is required to recover from a DNA damage-induced arrest in G1. a G1-synchronized RPE-1 cells were irradiated with a dose of 4 Gy; at the same time, BrdU and STLC were added and trypsinized at the indicated days after IR for flow cytometry analysis of BrdU-positive cells. Irradiated cells progressively entered into S-phase up to 6 days after IR. Depicted are the means and standard deviation of five independent experiments. b G1-synchronized RPE-Fucci cells were left unirradiated (−) or irradiated with a dose of 4 Gy (+); protein was harvested at the indicated hours post-damage (hpd) and analyzed by western blot. HSP90 was used as a loading control. IR induced a robust phosphorylation of CHK2 at S516 that decreased over time. c RPE-Fucci cells were trypsinized 40 hpd and arrested (expressing a red fluorofore) vs recovered (expressing a green fluorofore) cells were isolated using Fluorescence Activated Cell Sorting for subsequent western blot analysis. FACS plot indicates the gating windows of arrested (red) and recovered (green) cells. d Western blot analysis of arrested (A) vs recovered (R) cells 40 h after irradiation; pCHK2 S516 phosphorylation is lost in cells that recovered from IR