Figure S3.

FUS is required for efficient DSB repair and DNA damage foci formation upon genotoxic insult. (A) DDR activation in HeLa WT and FUS-KO cells upon ETO treatment. Cells were treated with 10 µM ETO for 1 h and were allowed to recover in ETO-free medium for 2 h (ETO release). Cells were collected at the indicated time points, lysed in the presence of phosphatase inhibitors, separated on a gradient SDS-PAGE, and processed for Western blotting (loading control: Actin). ATR and BRCA1 were probed on the same blot as the proteins shown in Fig. 2 B. MW, molecular weight. (B) DSB repair efficiency was quantified in U2OS cells containing a stably integrated NHEJ reporter system. Cells were silenced for FUS, KU80, or both. Right: Western blot demonstrating the silencing of the respective proteins. Data are presented as the mean ± SEM (experiments done in triplicate, with at least 10,000 cells analyzed per experiment). Statistics: one-way ANOVA, followed by Bonferroni post hoc test. *, P < 0.05. (C) DSB repair efficiency was quantified in U2OS cells containing a stably integrated HR reporter system. Cells were silenced for FUS, TOPBP1, or both. Right: Western blot demonstrating the silencing of the respective proteins. Statistical analysis as in A. ***, P < 0.001. (D) HeLa WT and FUS-KO cells were stained with γH2AX and counterstained with DAPI. Cells were treated with DMSO, ETO for 1 h, or ETO plus 2-h recovery from ETO treatment (ETO/2h). These are representative figures for graph shown in Fig. 2 C. Scale bar: 20 µm. (E) HeLa WT and FUS-KO cells were stained with 53BP1 and counterstained with DAPI. Cells were treated with DMSO, ETO for 1 h, or ETO plus 2-h recovery from ETO treatment (ETO/2h). These are representative figures for graph shown in Fig. 2 D. Scale bar: 20 µm. (F) 53BP1 expression is affected by neither KO of FUS nor by the ETO exposure. Loading control: Tubulin.