EdU and IdU incorporation assay

Cells were labeled with 10 μM EdU (Baseclick, BCK-EdU555) for 30 min and washed with fresh culturing medium. Cells were then treated or not treated with olaparib (3 μM) for 1 hour and labeled with 200 μM IdU (Sigma-Aldrich) thymidine analog for 30 min. Cells were fixed with 3% PFA for 10 min and treated with 2.5 M HCl to denature the DNA. Cells were washed three times with PBS and immunostained with anti-IdU antibodies. EdU incorporation was visualized by a Click-iT kit (Baseclick) according to the manufacturer’s instructions. Nuclei were stained with Hoechst. The EdU and IdU foci related to the replication fork movement were imaged with an LSM800 confocal setup with a Plan-Apochromat 20×/0.8 or a Plan-Apochromat 63×/1.4 oil objective and controlled by ZEN 2.3 software. Fluorescence excitation was performed using diode lasers at 405, 488, and 561 nm.