Fig. 3. NXF1 expression reverts Nsp1-mediated mRNA export block and reduction in mRNA levels.

(A) 293T cells were transfected with plasmids encoding GFP, GFP-Nsp1, or GFP-Nsp1 and Flag-NXF1 and then analyzed by RNA-FISH to detect poly(A) RNA and immunofluorescence to detect GFP, GFP-Nsp1, and Flag-NXF1. White arrowheads show examples for each condition. Scale bars, 10 μm. (B) Fluorescence intensity of total poly(A) RNA in individual cells is shown in the depicted conditions. (C) Fluorescence intensity of poly(A) in the nucleus and cytoplasm was determined, and the ratios of the nuclear-to-cytoplasmic signals for individual cells are shown for the indicated experimental condition. n = 3; ****P < 0.0001, ***P < 0.001, and **P < 0.01. (D) SK-N-SH cells were transfected with 3xFlag-Nsp1, and immunofluorescence microscopy was performed to detect Nsp1 and endogenous Nup358. n = 3. Scale bar, 5 μm. (E) Immunoprecipitation of Nsp1 followed by Western blot analysis shows Nsp1 interaction with certain nucleoporins, which is partially dependent on RNA.