Checkpoint maintenance is dependent on DNA end resection. (A) U2OS cells or U2OS cells stably expressing siRNA-resistant GFP-CtIP were transfected with the indicated siRNA for 2 d. Samples were fixed 8 h after IR and 6 h after nocodazole addition and then processed as in Fig. 3 A. For each sample, the H3Ser10p value was divided by the corresponding nocodazole (Noco) control to normalize for mitotic entry in the absence of IR. Data are the means of three independent experiments. Error bars represent SDs. WT, wild type. (B) U2OS cells or U2OS cells stably expressing siRNA-resistant GFP-CtIP were transfected for 2 d using the indicated siRNAs. The cells were harvested and processed for immunoblotting using the indicated antibodies. endo, endogenous. (C) U2OS cells were treated with DMSO or the ATM inhibitor (ATMi) 30 min before or 1 h after IR (5 Gy), as indicated. Nocodazole was applied 1.5 h after IR, and samples were fixed 6 h after IR. The bar chart shows the percentage of Histone H3-S10p–positive cells measured by flow cytometry and calculated as in A. Data are the means of three independent experiments. Error bars represent SDs. (D) U2OS cells were transfected with the indicated siRNA for 2 d, and samples were fixed at the indicated time points after IR and then processed as in A. Data are the means of three independent experiments. Error bars represent SDs. siUNC, Universal Negative Control siRNA. (E) Illustration of resection-independent checkpoint activation followed by resection-dependent maintenance of the ATR–CHK1-dependent S- and G2-phase checkpoints. Red circles indicate RPA. P, phosphorylation.