Fig. 2.

T457A has no effect on the efficiency of Polη to bypass CPDs. a 293T cells transfected with GFP-Polη were treated with Thiamet-G and glucose. Cells were irradiated with UVC (15 J m⁻²) and harvested 4 h later (top panel) or at the indicated time points (bottom panel). The cell lysates were immunoprecipitated with GFP-Trap A followed by immunoblotting with O-GlcNAc and GFP antibodies. Experiment was repeated three times. SE short exposure, LE long exposure. b 293T cells transfected with Flag-Polη were irradiated with UVC (15 J m⁻²) and harvested 4 h later. The cell lysates were immunoprecipitated and the relative IPed OGT was shown. Experiment was repeated twice. c U2OS cells transfected with indicated GFP-Polη constructs were UVC irradiated and incubated for 8 h. The proportions of GFP-Polη expressing cells with >30 foci were determined by counting at least 200 cells in each experiment. d Polη-deficient XP30RO cells infected with GFP, WT or T457A GFP-Polη lentivirus were selected by flow cytometry. The levels of Polη protein in stable clones were examined with anti-Polη. Tubulin: loading control. e Representative images of cells stained with CPDs (red) in ssDNA of nuclei (DAPI, blue) after UV irradiation. XP30RO cells stably expressing GFP, WT or T457A GFP-Polη were irradiated with 5 J m⁻² UVC and cultured for 4 h. The cells were treated with 1% Triton-X100 for 2 min prior to fixation. Scale bars: 10 μm. f Representative images of cells stained with DAPI and RPA2 after UV irradiation. XP30RO cells stably expressing GFP, WT or T457A GFP-Polη were irradiated with 5 J m⁻² UVC and further cultured. At the indicated time points, cells were treated with 0.5% Triton-X100 for 30 min followed by immunofluorescence. Scale bars: 10 μm. g Quantification of the percentage of cells with >10 RPA foci. For each cell line at each time point, at least 250 cells were counted. Data represent means ± SEM from three independent experiments. ns not significant