Endogenous FUS is partially mislocalized in patient fibroblasts with G504Wfs*12 and R495* mutations. a Primary fibroblasts cultures examined by confocal microscopy. A representative control image shows intense staining for FUS (green) in the nuclei (DAPI) and the stress granule markers eIF4G (red) in the cytoplasm. Patients with the G504Wfs*12 and R495* mutations near the NLS region also show that a majority of FUS protein in the nuclei with a slight increase of cytoplasmic FUS. In response to oxidative stress conditions, cytoplasmic FUS-positive inclusion bodies of G504Wfs*12 and R495* mutation co-localized with eIF4G stress granules (red). Cells were counter stained with the nuclear marker DAPI (blue). Scale bars = 25 μm. Bar graphs represent b the numbers of stress granules and c the numbers of FUS-positive stress granules (SGs). Data are from three experiments (the mean ± SEM, n = 20). One-way ANOVA followed by Tukey multiple comparisons test; **p < 0.001; N.S., not significant. d Cell fractionation analysis of cultured fibroblasts from ALS patients and controls showing an increased cytoplasmic expression of FUS in G504Wfs*12 and R495* patients compared with a representative control and Q519E patient. The upper band of FUS in the nucleus fraction of FUS (p.R495*) patient fibroblasts presumably an allele without a mutation and the lower band indicates the allele with the truncated R495* fragment. Lamin B2 and GAPDH are loading controls for the nuclear and cytoplasmic fractions, respectively. e HEK-293 cells were transfected with green fluorescent protein (GFP) wild-type FUS or FUS containing the ALS-associated mutations and treated with vehicle or 0.5 mM arsenite for 30 min. The cells were then processed for immunofluorescence analysis. Localization of GFP-tagged FUS wild type or the indicated FUS mutations (green), eIF4G stress granules (red) are shown. Cytosolic eIF4G co-localizes with FUS aggregates after oxidative stress. GFP (green) and eIF4G (red) show an increased overlap between mutant FUS (p.G504Wfs*12, p.R495*) and eIF4G as compared to wild-type FUS (WT) and eIF4G. Nuclei are shown by DAPI staining. Scale bars = 10 μm. f Rat E18 primary cortical neurons were cultured for 21 days and were transfected with constructs expressing wild-type FUS or ALS-associated mutants of FUS (green). After stress, redistribution of mutant FUS aggregates (green) into eIF4G (red) under oxidative stress is demonstrated. Nuclei are shown by DAPI staining. Scale bars = 25 μm.