NXF1 is present at the NPC independently of mRNA. (A) PolyA+ RNA detection by RNA FISH in U2OS cells. NXF1 (magenta) is detected in the NPCs while the mRNA (green) is not. The plot shows the intensity of both channels along the line in the merge. (B) PolyA+ RNA detection by RNA FISH in control cells and cells treated with DRB followed by RNase A digestion (RNase was added after permeabilization with 0.5% Triton, 2 min) and stained for NXF1. (C) Immunofluorescence of hnRNP A1, UAP56, CBP80, and NXF1 in control or DRB-treated U2OS cells. Decomposition of nucleoli is seen in the differential interference contrast (DIC) images indicating DRB transcription inhibition. (D) U2OS cell expressing the mutant Myc-NXF1-10RA protein that cannot bind mRNA (anti-Myc) and anti-Nup358 immunofluorescence for NPC detection. Cells were imaged at the middle (mid) or the top planes of the nucleus (top), and merged images show the presence of NXF1-10RA mutant in the NPCs. (E) NXF1 in control cells (top) or WGA-treated cells (bottom). Scale bars, 5 µm.