Fig 2: Transcriptional inhibition and hyperosmolarity induces FUS cytoplasmic translocation. (A) U87 cells were incubated with actinomycin D (ActD, 5 ug/ml), flavopiridol (FLV, 1 uM) or sorbitol (0.4 M) for 4 h, followed by paraformaldehyde fixation and FUS immunofluorescence (IF) using primary anti-FUS antibodies. Samples were washed, stained with secondary antibody, mounted and sealed for confocal imaging. (B) Cytoplasmic to nuclear FUS ratios were compared with the DMSO control. n = 24–33 cells were analyzed in each experimental condition. (***p = 0.0003, ****p < 0.0001) Mann–Whitney U-test. (C) U87 cells were incubated with 1 uM flavo for 4 h followed by FUS and TDP-43 IF. (D) Magnified images of FUS and TDP-43 co-staining. (E) Cytoplasmic to nuclear ratios were quantified for each protein in (C). n = 11–17 cells were analyzed in each experimental condition. (****p < 0.0001) Mann–Whitney U-test. (F) U87 cells were incubated with 1 uM flavo for 4 h followed with FUS and hnRNP C IF. (G) Cytoplasmic to nuclear ratios were quantified for each protein in (F). n = 15 ~ 18 cells were analyzed in each experimental condition. (****p < 0.0001) Mann–Whitney U-test.

Fig 3: FUS isoforms are differentially recognized by N- and C-terminal antibodies. Western blot analysis of whole-cell lysates of U87 cells transfected with FUS siRNA. FUS immunoreactive bands were blotted with antibodies recognizing either an (A) N- terminal (1–50 aa) or (B) C-terminal (500–526 aa) epitope. FL: Full-length FUS; NTF: N-terminal fragment; CTF (17): C-terminal fragment at 17 kDa; CTFs (25–50): C-terminal fragments distributed from 25 to 50 kDa; ns: non-specific band. Uncut nitrocellulose membrane was first blotted with anti-FUS antibody and HRP-conjugated secondary antibody followed by film exposure. The same membrane was then washed with 0.1% TBST and incubated with anti-GAPDH antibody. GAPDH bands were acquired using Bio-Rad ChemiDoc MP imaging system. Original blots are shown in the Expanded file 2.

Fig 4: Transcriptional inhibition induces FUS cytoplasmic translocation demonstrated by staining with multiple antibodies. (A, B) U87 cells were treated with flavo (1 uM, 4 h) followed by fixation and FUS IF. Each of the two anti-FUS antibodies recognizing the N-terminus (a, b) were mixed with one of the two anti-FUS antibodies recognizing the C-terminus (c, d) as indicated for FUS IF. The primary anti-FUS antibodies were distinguished by fluorescence-conjugated secondary antibodies recognizing different species (red: mouse; green: rabbit). Final concentration of each primary antibody was 2 ug/ml. (C) Enlarged images of insets indicated in panel A and B. (D) Cytoplasmic to nuclear FUS ratios were compared between flavo-treated (FLV) and untreated control (NT) cells using anti-FUS N-terminus and C-terminus antibody fluorescent intensities. n = 13–26 cells were analyzed in each experimental condition. (**p = 0.0088, ****p < 0.0001) Mann–Whitney U-test. Scale bars = 15 µm.

Fig 5: Nascent transcript levels are reduced in iPS cells derived from sporadic ALS patients Cumulative nascent transcript peak distribution of binding surface score of (A) total and (B) FUS-bound nascent transcripts in control (Ctrl) and sporadic ALS (sALS) iPSCs. (****p < 0.0001) Mann–Whitney U-test. (C, D) Mean expression level of (C) total and (D) FUS-bound nascent transcripts in control and sporadic ALS iPSCs. (****p < 0.0001) Mann–Whitney U-test.