Journal: Scientific Reports

Article Title: Nuclear RNA transcript levels modulate nucleocytoplasmic distribution of ALS/FTD-associated protein FUS

doi: 10.1038/s41598-022-12098-4

Figure Lengend Snippet: NXF1 and MTR4 knockdown, but not CRM1 inhibition, promotes FUS nuclear retention following transcriptional inhibition. ( A ) U87 cells were transfected with NXF1 siRNA for 48 h followed by poly(A +) RNA FISH and FUS IF. NT: No treatment; FLV: Flavo treatment (1 uM) for 4 h. Scale bar = 10 um. ( B ) Quantification of cytoplasmic to nuclear FUS ratios in siCtrl and siNXF1 transfected cells. n = 19–49 cells were analyzed in each experimental condition. (** p = 0.0033, **** p < 0.0001) Mann–Whitney U -test. ( C ) U87 cells were transfected with MTR4 siRNA for 72 h followed by poly(A +) RNA FISH and FUS IF. NT: No treatment; FLV: Flavo treatment (1 uM) for 4 h. Scale bar = 15 um. (D) Nuclear to cytoplasmic poly(A +) RNA ratios in siCtrl and siMTR4 transfected cells. n = 27–43 cells were analyzed in each experimental condition. (*** p = 0.0007, **** p < 0.0001) Mann–Whitney U -test. (E) Quantification of cytoplasmic to nuclear FUS ratio in siCtrl and siMTR4 transfected cells. n = 27–37 cells were analyzed in each experimental condition. (** p = 0.0015, *** p = 0.0001–0.0004, **** p < 0.0001) Mann–Whitney U -test. (F) U87 cells were treated with flavo (1 µM) and/or leptomycin B (LMB, 40 nM) for 4 h followed by FUS IF. Scale bars = 15 um. (G) Cytoplasmic to nuclear FUS ratios of flavo- and/or LMB-treated cells. n > 20 cells were analyzed in each experimental condition. (**** p < 0.0001) Mann–Whitney U -test.

Article Snippet: DNA damage was induced by doxorubicin (0.5 uM) for 24 h. To induce oxidative stress, sodium arsenite (500 uM) was added to growth media for 1 h. Osmotic stress was induced by 0.4 M sorbitol treatment for 6 h. Leptomycin B (LMB, Alomone Labs cat # L-500) was diluted in growth media to final concentrations of 10 to 40 nM and incubated with U87-MG cells from 2 to 6 h depending on the specific experiment.

Techniques: Inhibition, Transfection, MANN-WHITNEY