Expression of the FUS R495X mutant interferes with stress granule assembly in neuronal cells. (A) Exogenous human (h) FUS proteins are similar to each other and within 2-fold of endogenous mouse (m) FUS as determined by western blot and densitometry analyses. The ratio of hFUS/mFUS proteins is indicated for each cell line. (B) Immunofluorescence images of NSC-34 cells expressing untagged FUS (WT and R495X) either in the absence (−) or presence (+) of 0.25 mM sodium arsenite (SA) for 1 hr. Low magnification (40x) images of SA (+) treated cells using the anti-G3BP stress granule marker (green) show a greater number of stress granule-positive cells (denoted by arrows) in the FUS WT line compared to the FUS R495X line. High magnification (100X) images showing a single cell reveal the localization of FUS (red) and G3BP (note: the far red channel was used for detection of G3BP and the images are shown in green for clarity) under all conditions. DAPI (nuclei) is included in the overlay images. Note that in cells with stress granules, cytoplasmic R495X co-localizes to these structures as expected (see overlay image in bottom right). Scale bar = 20 and 5 μm, respectively, in low and high magnification images. (C) Quantitative analysis (as described in Figure 1B) of the percentage of NSC-34 cells with stress granules after 1 hr of sodium arsenite treatment revealed that expression of FUS R495X inhibited stress granule assembly compared to cells expressing FUS WT and the parent line. All error bars represent SEMs. Statistically significant differences were determined by one-way ANOVA and Tukey’s post-hoc test (****P < 0.0001, ***P < 0.001, **P < 0.01).