Fig. 7: The RINF168 SPaCR motif supports radio-resistance through suppression of 53BP1 accumulation. | Nature Communications

Fig. 7: The RINF168 SPaCR motif supports radio-resistance through suppression of 53BP1 accumulation.

From: PIN1-SUMO2/3 motif suppresses excessive RNF168 chromatin accumulation and ubiquitin signaling to promote IR resistance

Fig. 7

A Colony survival of U2OS cells depleted of RNF168 and complemented with myc-WT-RNF168, T208A, P209A or T208A/P209A variants after treatment with indicated doses of IR. The number of replicates (n) for each condition is mentioned in parentheses. Data is mean ± s.e.m. p-value compared to siNTC at 2 Gy IR, 7.52 × 10−7 for siRNF168, 0.91 for RNF168-WT, 4.7 × 10−7 for RNF168-T208A, 0.73 for RNF168-P209A and 0.03 for RNF168-T208A/P209A. Source data are provided as a Source Data file. B Colony survival of U2OS cells depleted of RNF168 and complemented with myc-WT-RNF168, T208A, K210R and P209A/K210R variants of myc-RNF168 after treatment with indicated doses of IR. The number of replicates (n) for each condition is mentioned in parentheses. Data is mean ± s.e.m. p-value compared to siNTC at 2 Gy IR, 7.52 × 10−7 for siRNF168, 0.91 for RNF168-WT, 4.7 × 10−7 for RNF168-T208A, 0.00001 for RNF168-K210R and 0.00002 for RNF168-P209A/K210R. Source data are provided as a Source Data file. C Western blot to show depletion of RNF168 and complementation of RNF168 variants for (A) & (B) (performed once). Source data are provided as a Source Data file. D BRCA1 assessment in U2OS expressing siRNA resistant myc-RNF168-WT or T208A, depleted of endogenous RNF168 and 53BP1 and treated with 2 Gy IR. 2 hrs post IR, cells were stained for BRCA1 and CENPF. Scale bars 10 µm. E Quantification of number BRCA1 foci from (D). Data is mean ± s.e.m, n = 77 cells for EV, 82 for myc-RNF168-WT, 92 for RNF168-T208A and 69 for RNF168-T208A+si53BP1. Source data are provided as a Source Data file. F Colony survival of U2OS cells depleted of RNF168 with or without si53BP1 and complemented with myc-WT-RNF168 or T208A after treatment with indicated doses of IR. n = 3. Data is mean ± s.e.m. p-value compared to siNTC at 2 Gy IR, 0.03 for siRNF168, 0.95 for RNF168-WT, 0.0058 for RNF168-T208A and 0.96 for RNF168-T208A + si53BP1. Source data are provided as a Source Data file. G Western blot to show depletion of RNF168, 53BP1 and complementation of RNF168 variants (performed once). Source data are provided as a Source Data file. H Illustration of the post-translational modifications at the RNF168 SPaCR ‘SUMO-PIN1-assisted Chromatin Regulator’ motif to regulate RNF168 dissociation from chromatin.

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