Fig. 2: UBE2D3 promotes NHEJ at uncapped telomeres and in a wider genomic context.
From: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168
a Quantification of chromosome fusions in TRF2ts cells transduced as indicated upon 24 h of telomere uncapping at 39 °C (n = 4 independent experiments; mean ± SEM; two-tailed Student’s t-test). b Quantification of chromosome fusions in control and UBE2D3-depleted SV40-immortalised wild-type (WT) MEFs, at 6 days after Trf2 shRNA expression (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). c Top: Quantification of chromosome fusions in control and UBE2D3-depleted HeLa cells expressing a doxycycline-inducible TRF2 shRNA (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Bottom: Immunoblotting for TRF2 and UBE2D3 in cells used for chromosome fusion analysis. The asterisk indicates TRF2. Representative blots from 3 independent experiments. d Top: Quantification of aneuploidy (>4N DNA content) based on DNA content analysis by flow cytometry of TRF2ts MEFs, transduced as indicated, subjected to telomere uncapping and stained with propidium iodide (n = 2 independent experiments). Bottom: Immunoblotting for UBE2D3 in cells used for aneuploidy assays. Representative blots from 2 independent experiments. e Telomeric G-overhang signal in control and UBE2D3-depleted TRF2ts MEFs at 32 °C and after 48 h of telomere uncapping. Left: Native conditions to detect single-strand (ss) TTAGGG repeats. Right: Denaturing conditions for total telomeric DNA. f Quantification of relative telomeric G-overhang signal (n = 2 independent experiments). g NHEJ-mediated repair in U2OS cells transduced as indicated and analysed for random plasmid integration. ShRNAs against the NHEJ promoting factors 53BP1 and RIF1 serve as positive controls (mean ± SEM of n = 3 independent experiments for 53BP1 sh and UBE2D3 sh2 and n = 2 independent experiments for RIF1 sh are shown). Statistical significance was calculated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. h Top: Survival assays of TRF2ts cells depleted for UBE2D3 with Ube2d3 shRNA1 and complemented with RNAi-resistant (RR) wild-type UBE2D3 (LZRS_UBE2D3_WT_RR) and UBE2D3 C85A (LZRS_UBE2D3_C85A_RR). Representative plates from 3 independent experiments. Bottom: Western blot analysis of Ube2d3 depletion, and UBE2D3 RR and UBE2D3 C85A RR expression in TRF2ts MEFs used in (h). Representative blots from 3 independent experiments. Ref reference. Source data are provided as a Source Data file.
