37 c heating stage

Camptothecin (Sigma-Aldrich) was added to cells for 1 hour at a final concentration of 1 µM. For IR treatments a Faxitron-CellRad (Faxitron Bioptics, LLC) was used. Localised lines of DNA-damage were induced by laser micro-irradiation, essentially as described previously53 (link),54 (link). Briefly, U2OS cells were plated on glass-bottom dishes (Willco-Wells), treated with the indicated siRNAs or drugs, and pre-sensitised with 10 µM BrdU (Sigma-Aldrich) in phenol red-free medium (Invitrogen) for ~48 hours at 37°C. Subsequent exposure to a laser beam was performed using a FluoView 1000 confocal microscope (Olympus) equipped with a 37°C heating stage (Ibidi) and a 405 nm laser diode (6 mW) focused through a 60× UPlanSApo/1.35 oil objective and resulting in a spot size of 0.5-1 µm. 250 ms laser beam exposure times (fast scanning mode) were used at a setting of 0.4 mW output (50 scans) to yield pre-sensitisation-dependent DNA-damage, restricted to laser tracks without detectable cytotoxicity. For experiments involving CtIP, a laser power of 0.20-0.25 mW output was used, which resulted specifically in its recruitment to laser tracks in Cyclin A (CycA)-positive S/G2 but not in CycA-negative G1 cells. Cells were laser micro-irradiated for ~20 minutes and left to recover under standard cell culture conditions for 1-2 hours before fixing and staining.