FIGURE 2:

Nuclear translocation of wild-type and mutant FUS is increased in RALY knockout cells. (A) Immunofluorescence of RALY KO and control HeLa cells transfected with HA-tagged FUS constructs and treated with arsenite. Cells were stained with anti-HA and anti-PABP1 antibodies and then detected with Alexa Fluor 594- and Alexa Fluor 488–conjugated secondary antibodies, respectively. The scale bar corresponds to 40 μm. (B) The graphs report the quantification of nucleus/cytoplasm signal of HA staining (corresponding to transfected FUS constructs), obtained by high-content image analysis, in untreated (top graph) and arsenite-treated HeLa cells (bottom graph). Bars indicate means ± SEM of five replicates, and p values were calculated with unpaired two-tailed Student’s t test to compare RALY KO to control cells (*p < 0.05; **p < 0.01; ***p < 0.001). (C) The graph reports the quantification of FUS-HA number of spots per cell, obtained by high-content image analysis. Spots, induced by arsenite treatment, were detected with PABP1 staining and then analyzed for HA-positive staining. Bars indicate means ± SEM of five replicates, and p values were calculated with unpaired two-tailed Student’s t test to compare RALY KO to control cells (**p < 0.01; ***p < 0.001).