Immunofluorescence Staining for PCNA and SIVA1

Indirect immunofluorescence was performed as previously described (Huang et al., 2009 (link); Liu et al., 2010 (link); Wan et al., 2013 (link)). HeLa or XP30RO (gift from C. Guo, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China) cells cultured on coverslips were treated with 50 J/m² UV and recovered for the indicated times. Cells were then washed with PBS, preextracted with buffer containing 0.5% Triton X-100 for 5 min, and fixed with 3% paraformaldehyde for 10 min at room temperature. For PCNA and SIVA1 coimmunofluorescence staining, HeLa cells were denatured by 2.5 M HCl for 1 h at room temperature after 3% paraformaldehyde fixation. Cells were then incubated in primary antibody for 30 min at room temperature. After three 5-min washes with PBS, secondary antibody was added at room temperature for 30 min. Cells were then stained with DAPI to visualize nuclear DNA. Images were captured with use of a fluorescence microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor 60× oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ²; Photometrics). Images were captured using NIS-Elements basic research imaging software (Nikon) and analyzed using Photoshop CS3 (Adobe).