Loss of FUS results in accumulation of DNA damage and sensitization to genotoxic insult in HeLa cells. (A) Total extracts of WT and FUS-KO HeLa cells were analyzed by Western blotting with anti-FUS and anti-γH2AX antibodies (loading control: Tubulin). FUS-KO cells display an 8.1-fold increase in the level of endogenous γH2AX in comparison to WT cells. (B) Representative confocal micrographs of γH2AX foci in WT and FUS-KO HeLa cells. Scale bar: 20 µm. Cropped single cells are enlarged 2× (scale bar: 5 µm). (C) Quantification of γH2AX foci. The number of foci per nucleus was counted using ImageJ. WT cells have an average of 1.02 foci per cell, compared with 1.82 in FUS-KO cells. Data are from two biological replicates (170 cells each). Statistics: Student’s t test. (D) HeLa FUS-KO cells were transiently transfected with a plasmid expressing FUS-Flag and stained with anti-Flag and anti-γH2AX. Foci were quantified by ImageJ. Data are from two biological replicates (65 cells each; only transfected cells were included). Statistics: Student’s t test. (E) HeLa WT and FUS-KO cell viability assessed by Trypan blue staining upon treatment with CPT (0.1 or 0.5 µM) or ETO (0.5 or 1 µM). Percentage survival was calculated by normalizing the number of surviving cells by their respective DMSO group. Statistics: two-way ANOVA, Bonferroni post hoc test. In all panels: *, P < 0.05; **, P < 0.01; ***, P < 0.001.