Figure 4.

SART3 is required for optimal cellular response after UV treatment. (A and B) SART3 deficiency exhibits significantly increased levels of CPD lesions. U2OS cells stably expressing either GFP or GFP-SART3 were transfected with siNC or siSART3, siPolη. 72 h later, the cells were irradiated with 10 J/m² UVC, further incubated for 2 h. Then the cells were permeabilized and fixed, immunostained with anti-CPD antibody and mounted with DAPI. (A) Data represent means ± SEM from three independent experiments. More than 200 cells were counted. (B) Knockdown efficiency was checked by immunoblotting. (C) SART3 ablation increases UV-induced mutation frequency. 293T cells were transfected with siNC or siRNAs of SART3 twice. Forty-eight hours after the first transfection, cells were transfected with UVC-irradiated (400 J/m²) pSP189 reporter plasmids. 48 h later, pSP189 plasmids were extracted from 293T cells and digested with Dpn1, followed by transformed into the MBM7070 bacterial strain for mutation frequency analysis. Data represent means ± SEM from three independent experiments. Statistical analyses were performed using a two-tailed Student's t-test. Knockdown efficiency was confirmed by immunoblotting. (D and E) The depletion of SART3 exhibits hypersensitivity to UV irradiation. Cell survival assays in SART3-depleted or Polη-depleted cells after UV exposure. SiNC, siSART3 or siPolη transfected cells were irradiated with indicated dosage of UVC, which were permitted to grow for 14 days before being counted. (D) Experiments were performed in triplicate. Error bars represent standard error. (E) Knockdown efficiency was confirmed by immunoblotting. (F) Percentage of cells containing one or more micronuclei in SART3 knockdown cells significantly increases under both basal and UV irradiation conditions. U2OS cells stably expressing either GFP or GFP-SART3 were transfected with siNC or siSART3, followed by 3 J/m² UVC exposure or not, then further incubated with 6 μg/ml CB for 48 h. Cells with micronuclei were counted. Data represent means ± SEM from three independent experiments.