CtIP is critical for IR-induced checkpoint maintenance. (A) U2OS cells or U2OS cells stably expressing siRNA-resistant GFP-CtIP were transfected with the indicated siRNA for 2 d, and samples were fixed at the indicated time points after IR. Nocodazole (Noco) was added 2 h after IR. The bar chart shows the percentage of Histone H3-Ser10p–positive cells measured by flow cytometry. Data are the means of three independent experiments. Error bars represent SDs. The inset blot shows expression levels of GFP-CtIP and endogenous (endo) CtIP in the used cells lines. siUNC, Universal Negative Control siRNA. (B) RDS assay was performed using U2OS cells or U2OS cells stably expressing siRNA-resistant GFP-CtIP. Cells were siRNA transfected for 2 d with the indicated siRNAs and subjected to RDS assay. Data are the means of three independent experiments. Error bars represent SDs. (C) U2OS cells were seeded on coverslips and transfected for 2 d with the indicated siRNAs. The samples were treated with IR and nocodazole, as indicated, followed by fixation. Cells were stained for Histone H3-S10p and γH2AX, and images were acquired using epifluorescence microscopy. Bar, 10 µm. (D) U2OS cells were treated as in Fig. S2 D. The bar chart shows the percentage of Histone H3-S10p–positive cells with more than five γH2AX foci for a given treatment. Data are the means of three independent experiments. For each sample, at least 50 Histone H3-S10p–positive cells were counted. Error bars represent SDs.