Loss of FUS results in accumulation of DNA damage and sensitization to genotoxic insult in SH-SY5Y cells. (A) Total extracts of WT and FUS-KO SH-SY5Y cells were analyzed by Western blotting with anti-FUS and anti-γH2AX antibodies. FUS-KO cells display a 2.6-fold increase in the level of endogenous γH2AX in comparison to WT cells. Tubulin was used as loading control. MW, molecular weight. (B) Representative confocal micrographs of γH2AX foci in WT and FUS-KO SH-SY5Y cells. Scale bar: 20 µm. Cropped single cells are enlarged 2× (scale bar: 5 µm). (C) Quantification of B. The number of foci per nucleus was counted using ImageJ and plotted as a violin plot. Data from two biological replicates, with 170 cells per replicate. The average foci number in WT cells is 1.01, compared with 1.71 in FUS-KO cells. Statistics: Student’s t test (***, P < 0.001). (D) SH.SY5Y WT and FUS-KO cell viability assessed by Trypan blue staining upon treatment with increasing concentrations of CPT (0.1 or 0.5 µM) or ETO (0.5 or 1 µM). Statistics: two-way ANOVA followed by Bonferroni post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).