IF was performed as previously described (Leung et al. 2014 (link)). Briefly, U2OS cells expressing shGFP or shFKBP25 were directly seeded on coverslips for overnight incubation. For Etopside damage, cells were treated with 100 μmol/L Etopside for 20 mins, and then washed and incubated for 2 h. For the ionizing radiation experiments, cells were treated with 5 Gy delivered by a Faxitron X-Ray machine. After IR, cells were incubated for 2 h. After the indicated treatment, cells were pre-extracted with cytoskeletal (CSK) buffer for 5 min on ice, fixed with 2% (v/v) formalin for 15 min at room temperature, and blocked with PBS containing 3% bovine serum albumin (BSA). After blocking, the cells were incubated with primary antibody overnight. After 3× PBS washes, the cells were incubated with secondary antibody for 1 h at room temperature. Primary antibody used was RAD51 (ab133534; Abcam). The secondary antibody used was Alex Fluor 488 goat anti-mouse IgG (Invitrogen). After slide preparation, imaging was processed and analyzed with the Z-stacked setting using the FV10-ASW3.1 software on a Fluoview 1000 confocal microscope (Olympus).