Figure 3.

SART3 regulates UV-induced RPA focus formation and single-stranded DNA generation. (A and B) SART3 ablation attenuates RPA focus formation after UV irradiation. U2OS cells stably expressing either GFP or GFP-SART3 were transfected with siNC or siSART3 and incubated for 72 h. Then the cells were irradiated with 15 J/m² UVC and further cultured. (A) Representative images of cells stained with DAPI or antibody against RPA32 after UV irradiation. The cells were permeabilized with 0.5% Triton X-100 for 30 min and fixed, immunostained with anti-RPA32 antibody and mounted with DAPI. (B) Quantification of the percentage of cells with RPA32 foci. The proportion of cells with more than 10 RPA32 foci were measured, at least 200 cells were counted. Data represent means ± SEM from three independent experiments. Western blots showed the knockdown efficiency in cells. (C) Depletion of SART3 reduces the level of RPA32 on chromatin. U2OS cells stably expressing either GFP or GFP-SART3 were transfected with siNC or siSART3, and irradiated with 15 J/m² UVC, repaired for 4 h. The triton-insoluble fractions (TIF) and whole-cell lysates (WCL) were harvested and analyzed with the indicated antibodies. (D and E) SART3 deficiency displays an obvious reduction in ssDNA formation. U2OS cells stably expressing either GFP or GFP-SART3 were transfected with siNC or siSART3 and incubated overnight, then the cells were labeled with 10 μM BrdU for 48 h, followed by 20 J/m² UVC irradiation, further incubated for 2 h. The cells were permeabilized with 0.5% Triton X-100 for 5 min and fixed, immunostained with anti-BrdU antibody. Representative images are shown (D). (E) The percentage of cells with more than 10 BrdU foci was quantified (at least 200 cells were counted). Data represent means ± SEM from three independent experiments. Western blots showed the knockdown efficiency of siRNA in cells.