Fig. 6

T457 affects Polη TLS efficiency after cisplatin treatment. a 293T cells transfected with Flag-Polη were treated with CDDP (10 µg ml⁻¹) for 2 h and changed to drug-free media for 2 h. The cell lysates were harvested for immunoprecipitation assay as in Fig. 1e. b 293T cells transfected with GFP-Polη were treated with Thiamet-G and glucose. Cells were treated with UVC (15 J m⁻²) irradiation or cisplatin (10 µg ml⁻¹) and harvested at 4 or 2 h later, respectively. The cell lysates were immunoprecipitated with GFP-Trap A and analyzed with anti-O-GlcNAc and anti-GFP antibodies. c XP30RO cells stably expressing GFP, WT, or T457A GFP-Polη were treated with cisplatin for 24 h and further incubated for 7–10 days. Cell survival was analyzed as in Fig. 3f. d U2OS cells transfected with WT or T457A GFP-Polη were treated with cisplatin (5 µg ml⁻¹) for 2 h followed by incubation with fresh media. At the indicated time points, the proportion of GFP-Polη expressing cells with >30 foci was determined. e Representative images of cells stained with DAPI and RPA2 after cisplatin treatment. XP30RO cells stably expressing GFP, WT, or T457A GFP-Polη were treated with cisplatin for 24 h and further cultured. At the indicated time points, cells were immunostained with anti-RPA2 antibody. Scale bars: 10 μm. f Quantification of the percentage of cells with >10 RPA2 foci. g Scheme of experimental design for replication fork progression in XP30RO cells stably expressing GFP, WT, or T457A GFP-Polη. After cisplatin (1 µg ml⁻¹) treatment for 24 h, representative DNA fibers were shown. h Median lengths of nascent replication tracts (labled with CIdU) were given. Data represent means ± SEM from three independent experiments. ns not significant