HEK293 cells containing a stably integrated copy of the EJ5‐GFP reporter for NHEJ were depleted of ataxin‐3 or MDC1 and repair of I‐SceI‐induced DSBs by NHEJ was quantified. Data are presented as mean ± SEM from three independent experiments. **P ≤ 0.01; ***P ≤ 0.001 (one‐way ANOVA test).
Random plasmid integration assay. U2OS cells were transfected with indicated siRNAs and with linearized pEGFP‐C1 plasmid DNA. After 5 days, cells were counted and re‐seeded in medium containing or lacking G418. Colonies were counted on day 15. Data are presented as mean ± SEM from two independent experiments. ***P ≤ 0.001; ****P ≤ 0.0001 (one‐way ANOVA test).
U2OS cells were depleted of ataxin‐3 or MDC1, subjected to laser micro‐irradiation and immunostained for XRCC4 and γH2AX. The relative increase in XRCC4 at laser‐induced DNA damage was quantified. Data are presented as mean ± SEM from two independent experiments. **P ≤ 0.01; ****P ≤ 0.0001 (Kruskal–Wallis test).
Parental U2OS cells or U2OS cells stably expressing GFP‐ataxin‐3 or GFP‐ataxin‐3C14A were depleted of ataxin‐3 (siATX3‐1). After micro‐irradiation, cells were immunostained for XRCC4. The levels of XRCC4 at sites of laser‐induced DNA damage were quantified. Data are presented as mean ± SEM from two independent experiments. **P ≤ 0.01; ****P ≤ 0.0001 (Kruskal–Wallis test). ns, non‐significant.
As in (C) but cells were transfected with siRNAs targeting ataxin‐3 or RNF4. Data are presented as mean ± SEM from two independent experiments. ****P ≤ 0.0001 (Kruskal–Wallis test).
For survival experiments, VH10‐SV40 cells were transfected with indicated siRNAs, seeded at low density and exposed to the indicated doses of ionizing radiation. Cells were incubated for 7 days and stained with methylene blue. Colonies of more than 10 cells were scored. Data are presented as mean ± SEM from two independent experiments.
Data information: Scale bars, 5 μm.
