Fig. 6.

Nup93 depletion reduces nuclear import but does not affect nuclear export. a A representative image of nuclear import assay performed using GR2-GFP2-M9 construct transfected in cells treated independently with LacZ and siNup93. To induce nuclear import of GR2-GFP2-M9 fusion protein, cells were treated with dexamethasone (Dex) (5 µM) for 30 min, white arrowhead indicates absence of cytoplasmic GFP in LacZ + Dex and residual cytoplasmic GFP in Nup93 Kd + Dex. Scale bar ~10 µm. b A representative image of Poly(A) RNA FISH performed using FAM-labeled oligo(dT) probe (green) in siNeg, Nup93 Kd, Nup188 Kd, Nup205 Kd and Nup98 Kd, scale bar ~10 µm, white arrowhead indicates Poly(A) RNA foci in the nucleus. Nuclear boundary is marked by dotted line in enlarged panel. Nup98 enlarged panel shows both nuclear and cell boundary with white dotted line. c Nuclear/cytoplasmic (N/C) ratio of GR2-GFP2-M9 was determined by quantifying its relative fluorescence intensity in the nucleus and cytoplasm. Scatter plot of GFP signals expressed as nuclear-to-cytoplasmic ratios from LacZ (n = 60 cells), Nup93 Kd (n = 57), Nup188 Kd (n = 60), Nup205 Kd (n = 59) and Nup98 Kd (n = 60), data from 2 independent biological replicates (****p < 0.0001). d Poly(A) RNA distribution was determined by quantifying its fluorescence intensity in the nucleus and cytoplasm. Scatter plot of Poly(A) signals expressed as nuclear (N)-to-cytoplasmic (C) ratios from siNeg (n = 127 cells), Nup93 Kd (n = 158), Nup188 Kd (n = 288), Nup205 Kd (n = 288); N/C ratio was not significant (ns) when compared to siNeg (p > 0.05), while Nup98 Kd (n = 97) shows a relatively higher nuclear-to-cytoplasmic ratio (N/C ratio) of Poly(A) signals (***p = 0.0017). Two independent biological replicates for siNeg, Nup93 Kd and Nup98 Kd. Data from a single experiment for Nup188 Kd and Nup205 Kd. Horizontal line represents median, p values obtained from Mann–Whitney U test. Nuclear transport of Poly(A) RNA was unaffected in Nup93-, Nup188- or Nup205- depleted cells