Fig. 6: T208A-RNF168 mutant drives increased chromatin ubiquitination, increased 53BP1 accumulation but reduced BRCA1 accumulation.

A Western blot of chromatin fraction for H2AK15ub and H2A from cells expressing siRNA-resistant myc-RNF168-WT or T208A. Endogenous RNF168 is depleted by siRNA. Cells were either left untreated or irradiated by 10 Gy IR and fractionated 1 hr later (Representative of 2 repeats). Source data are provided as a Source Data file. B U2OS cells treated with non-targeting siRNA or RNF168 siRNA and expressing siRNA-resistant myc-WT-RNF168 or myc-T208A-RNF168 were either untreated (left) or treated with 2 Gy of IR (right) and stained for 53BP1 and γH2AX. Scale bars 10 µm. C Quantification of 53BP1foci intensity from untreated cells. Data is mean ± s.e.m, n = 150 for siNTC, 100 for siRNF168, 140 for WT-RNF168 and 124 for T208A-RNF168. Source data are provided as a Source Data file. D Quantification of 53BP1 foci intensity from irradiated cells. Data is mean ± s.e.m, n = 177 for siNTC, 104 for siRNF168, 133 for RNF168-WT and 158 for RNF168-T208A. Source data are provided as a Source Data file. E BRCA1 assessment in U2OS expressing siRNA resistant myc-RNF168-WT, T208A or K210R, depleted of endogenous RNF168 and treated with 2 Gy IR. 2 hrs post IR, cells were stained for BRCA1 and CENPF. Scale bars 10 µm. F Quantification of number BRCA1 foci from (E). Data is mean ± s.e.m, n = 143 cells for siNTC, 153 for siRNF168, 147 for RNF168-WT, 129 for RNF168-T208A and 136 for RNF168-K210R. Source data are provided as a Source Data file. G Quantification of number RAD51 foci in U2OS expressing siRNA-resistant myc-RNF168-WT, T208A or K210R mutants, depleted of endogenous RNF168 and treated with 2 Gy IR. 2 hrs post IR, cells were stained for RAD51 and CENPF (representative images in Supplementary Fig. 6D). Data is mean ± s.e.m, n = 132 cells for siNTC, 207 for siRNF168, 195 for RNF168-WT, 297 for RNF168-T208A and 207 for RNF168-K210R. Source data are provided as a Source Data file.