Fig. 2.

S317 is required for the G₂/M checkpoint in response to IR. (A and B) Activation of the G₂/M checkpoint. Parental DLD-1 and Chk1 monoallelic cells DLD-Chk1^WT and DLD-Chk1^S317A were incubated in the presence of nocodazole (0.2 μg/ml) for the times indicated. Cells were untreated (A) or treated with 12 Gy IR (B). Cells were fixed and stained with Hoechst 33258. The mitotic index was determined by fluorescence microscopy. Approximately 200 cells were counted for each time point. (C) Degradation of Cdc25A. Following exposure to 12 Gy IR, monoallelic cells DLD-Chk1^WT and DLD-Chk1^S317A were lysed and probed with anti-Cdc25A antibody. α-Tubulin was assessed as a loading control. (D) Clonogenic survival following IR treatment. Cells were plated at low density and treated with the indicated doses of IR. Colony formation was assessed 14 d after treatment. Surviving fraction was determined after normalization to untreated controls. Each data point was derived from three independent plates.