Removal of NXF1 from NPCs. (A) U2OS cells were transfected with either GFP-HoxA9 (control) or GFP-2xGLFG-HoxA9 and immunostained with antibodies to NXF1 and Nup153. Overexpressed FG domains dissociated NXF1 (but not Nup153) from the NPC (arrow). Scale bar, 5 µm. (B) U2OS cells transfected with either GFP-HoxA1 (as control) or GFP-2xGLFG-HoxA9 were stained for PolyA+ RNA FISH and immunostained with anti-NXF1. Arrows point at transfected cells, indicating that overexpression of FG domains removes NXF1 from the NPC and inhibits mRNA export. Scale bar, 20 µm. (C) U2OS cells stably expressing YFP-MS2–tagged Cerulean-minidystrophin-MS2 mRNPs were transfected with mCerulean-2xGLFG-HoxA9 and induced to transcribe (4 h). Left: Frame from a representative live-cell video showing single mRNPs (green dots; large dots are sites of transcription). Right: Movie average time projection showing the static mRNPs (black dots). Number of static mRNPs in the nuclear envelope was calculated as in Fig. 1 (mRNPs tracked: 1,558 mRNPs, 22 cells). Measurements were performed in three independent experiments. Scale bar, 10 µm. (D) Average time projection (Video 9) showing an mRNP (green) stably inserted within the POM121-Cherry–tagged signal (NPCs, red) in a cell expressing mCerulean-2xGLFG-HoxA9. Scale bar, 0.5 µm.