Figure 6. NUP93 binds with Importin7, and its knockdown inhibits and overexpression enhances nuclear translocation of SMAD and ERK.

(A) Left and middle: confocal microscopy of cells transfected with the indicated siRNAs and probed for Importin7 and NUP93. Scale bars, 10 μm. Right: HEK293 T cells (8 × 10⁶) were co-transfected with Importin7 and NUP93 plasmids. EGF treatment (10 ng/mL; 7 min) was applied, 48 h later, on one plate. Cleared extracts were directly resolved or subjected to immunoprecipitation (IP) prior to immunoblotting (IB). The experiments were repeated at least twice. Scale bar, 10 μm.

(B and C) MCF10A cells expressing shNUP93, or control shRNA (SC), were stimulated with TGFB2 (B) or EGF (C). Fixed cells were stained for SMAD2/3 or pERK1/2. Shown are confocal images and quantifications of translocation that were performed in triplicates (averages ± SEM). The experiment was repeated twice. Scale bars, 10 μm.

(D) MCF10A cells pre-stimulated with EGF, as in (C), were fractionated. Lactate dehydrogenase and histone-3 were used as markers. CE and NE, cytoplasmic and nuclear extracts. The experiment was repeated thrice.

(E) MCF10A cells inducibly overexpressing NUP93 were induced and stimulated with EGF. Fixed cells were probed for pERK1/2 (left) and total ERK1/2 (right). The histograms show quantification of protein localization (means of triplicates ± SEM). The experiment was repeated twice.

(F) MDA-MB-231 cells overexpressing a Tet-inducible NUP93 were injected into the fat pad of NOG female mice. Metastasis to lungs was confirmed 15 days later. Thereafter, tetracycline was added to the drinking water and mice were sacrificed 2 weeks later. Deparaffinized lung slices were probed for pERK1/2. Mean fluorescence intensity (MFI) was calculated from eight areas of each slide. The experiment was repeated twice.

****p ≤ 0.0001 in (B), (E), and (F).