RAD51C assembly at break sites depends on RPA. (A) HeLa cells were irradiated (10 Gy) and fixed 2 h later or treated with 2 mM HU for 17 h, released into fresh media, and fixed 4 h after release. Cells were stained with anti-RAD51C (red) and anti-RPA antibodies (green). Nuclei were visualized with DAPI (blue). (B) Control or RAD51C siRNA was cotransfected with a GFP-expressing vector. U2OS cells were irradiated 48 h after transfection, fixed after 2 h, and stained with anti-RPA and anti-RAD51 antibodies (red). (C) siRNA-mediated reduction of RPA expression. Cells were transfected with control or RPA siRNA and irradiated 48 h after transfection. Cell extracts were analyzed by Western blotting against RPA and tubulin. (D) Control or RPA siRNA was cotransfected with a GFP-expressing vector into U2OS cells. Cells were irradiated 48 h after transfection, fixed after 2 h, and stained against RAD51C. Bars, 10 µm.