Checkpoint initiation and CtIP-dependent DNA end resection are temporally separated. (A) U2OS cells were either left untreated or treated with 1 µM CPT for the indicated time. Cells were harvested and processed for immunoblotting with the indicated antibodies. (B) U2OS cells were seeded on coverslips and incubated with BrdU for 24 h. Cells were treated with 1 µM CPT for the indicated time, fixed, and stained with BrdU and 53BP1 antibodies under native conditions. Pictures were acquired using epifluorescence microscopy. Bar, 10 µm. (C) U2OS cells were transfected for 2 d with the indicated siRNAs. Cells were treated with 1 µM CPT for the indicated time and then processed as in A. Numbers represent quantification of phosphorylated CHK1. siUNC, Universal Negative Control siRNA. (D) U2OS cells were transfected for 2 d with the indicated siRNAs. The cells were treated with 0.1 µM or 1 µM CPT and then processed as in A. Numbers are as in C.