Fig. 4.

A distribution of NPCs is affected in TPR-depleted HeLa cells (shTPR HeLa cell line). a–c The distribution of NPCs in control HeLa cells as viewed by immunostaining with anti-Nup62 antibody and wide-field Leica DM6000 microscope. a Homogenous distribution. b Small NPC-free zones. c Large NPC-free zones comprehend more than 1/10 of nuclear surface. d The ratio of homogenous distribution, small and large NPC-free zones in control wild-type S-G2 cells (wt), in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR cells). In TPR-depleted cells, the number of nuclei with NPC-free zones increased significantly (χ² = 11.41, df = 2, P < 0.001, Chi squared test), **P < 0.01, ***P < 0.001, n.s. not significant. e The ratio of homogenous, small and large NPC-free zones in control wild type cells (wt) and in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR) synchronized in G1/S and in G2 phase of the cell cycle. The number of NPC-free zones significantly increased in TPR-depleted cells (***P < 0.001, n.s. not significant). f–i Recruitment of nucleoporins Nup107, Nup133 and central FG-Nups into NPC-free zones is impaired in TPR-depleted cells. f The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with Nup153 antibody (χ² = 7.08, df = 2, P = 0.029). g The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells transfected with GFP–Nup107 construct (χ² = 9.713, df = 2, P = 0.008). h The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells overexpressing GFP–Nup133 (χ² = 23.855, df = 2, P < 0.001). i The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with antibody recognizing central FG-Nups (χ² = 138.5, df = 2, P < 0.0001)