Fig. 4: UBE2D3 prevents RNF168 hyperaccumulation through ubiquitination and degradation of RNF168 and thereby facilitates telomere NHEJ.

a Immunoblots to evaluate RNF168 protein stability in control and Ube2d3 shRNA2 transduced cells, cultured in the presence of 50 μg/ml cycloheximide (CHX). Representative blots from 5 independent experiments. b Quantification of cycloheximide experiments as in a (n = 5 independent experiments; mean ± SEM; two-tailed Student’s t-test). Significance is shown for Control vs. UBE2D3 sh2: *p = 0.0488 (8 h), ns p = 0.0524 (16 h), *p = 0.0307 (24 h). c Immunoblots for RNF168 in control and UBE2D3-depleted TRF2ts cells at 32 °C or upon telomere uncapping and treated for 4 h with 10 μM MG132. Representative blots of two independent experiments. d Immunoblots for GFP-RNF168 in control and UBE2D3-depleted TRF2ts cells at 32 °C or upon telomere uncapping and treated for 4 h with 10 μM MG132. Representative blots of two independent experiments. The asterisk annotates the band for GFP-RNF168. e HeLa cells with or without expression of His-Ubiquitin were transduced with control or two independent lentiviral shRNAs targeting UBE2D3. His-ubiquitin conjugates were purified and analysed by immunoblotting. Representative of 3 independent experiments. See Supplementary Fig. 6b for UBE2D3 levels in input samples. f Quantification of polyubiquitinated GFP-RNF168 (Ubⁿ) immunoprecipitated from control or UBE2D3-depleted 293T cells, transfected with HA-Ubiquitin and GFP or GFP-RNF168 (n = 2 independent experiments). The eluted poly-HA-Ub signal was corrected over the eluted GFP-RNF168 signal. Corresponding blots in Supplementary Fig. 6e, f. g Quantification of chromosome fusions in control, Ube2d3 shRNA1 or GFP-RNF168 transduced TRF2ts cells upon 24 h of telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). h Immunoblotting for GFP-RNF168 expression and Ube2d3 depletion in TRF2ts cells used in (g). Asterisk indicates GFP-RNF168. i Immunoblotting for RNF168 in TRF2ts cells transduced as indicated upon 24 h of telomere uncapping at 39 °C. Representative blots from 3 independent experiments. j Chromosome fusions in TRF2ts MEFs transduced with indicated shRNAs upon 24 h of telomere uncapping (n = 3 independent experiments; mean ± SEM; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). Ref reference. Source data are provided as a Source Data file.