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Fig. 4 | BMC Biology

Fig. 4

From: Sustained CHK2 activity, but not ATM activity, is critical to maintain a G1 arrest after DNA damage in untransformed cells

Fig. 4

CHK2 self-activity after DNA damage enables G1 arrest establishment independently of ATM. a Left panel: experimental setup. G1-synchronized RPE-1 cells were irradiated with a dose of 4 Gy, and 1 h after cells were either left untreated (−ATMi) or treated with ATM inhibitor (+ATMi); protein was harvested at the indicated timepoints. Right panel: western blot analysis of protein extracts. b Upper panel: experimental setup. G1-synchronized RPE-1 cells were irradiated with a dose of 4 Gy, and 1 h after IR inhibitors for either CHK2 alone (CHK2i, blue line) or CHK2 and ATM (CHK2iATMi, blue and red line) were added; 1 h later (2 h timepoint) inhibitors were washed out, and cells were incubated in the absence (−ATMi, black line) or presence (+ATMi, red line) of ATM inhibitor for two additional hours (4 h timepoint). Lower panel: western blot analysis of protein extracts from 2 h and 4 h timepoints. c Cells treated as in b were cultured for 40 h with BrdU/STLC in the presence or absence of ATMi during inhibitor release and collected by trypsinization for flow cytometry analysis; a condition where inhibitors were added at the time of IR (0 h) and maintained until trypsinization was used as a control for G1 recovery. Depicted are the means and standard deviation of three independent experiments. d Upper panel: the experiment in b was repeated with an CHK2i or CHK2iATMi pulse of 3 h and 1 h release in the absence of inhibitors (−Inhibitors, black line), presence of ATM inhibitor (+ATMi, red line) or presence of ATM, ATR, and DNA-PKcs inhibitors (+ATMi+ATRi+DNAPKi, purple line). Lower panel: western blot analysis of protein extracts of 4 and 5 h post-damage (hpd). e Cell cycle analysis of cells treated as in d, and cultured in the presence of BrdU/STLC during inhibitor release

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