Figure 2. Nup153 is necessary for Repo-Man targeting to the nuclear periphery.

(a) Anaphase from HeLa stable cell line expressing GFP:Repo-Man^N (N terminus Repo-Man), stained for Nup153 (red). Late anaphase showing Repo-Man accumulation at the chromosome periphery and co-localization with Nup153. (b) HeLa cells transfected with Control (1) or Nup153² RNAi oligo (2) and stained for endogenous Nup153 (red) and endogenous Repo-Man (green). Zoom-in image of Nup153 and Repo-Man at the chromosome periphery in control cells (3). Scale bar, 5 μm. (c) Quantification (line profile analysis) of Repo-Man enrichment at the chromosome periphery. (d) Interphase nucleus of GFP: Repo-Man^N HeLa stable cell line showing the nuclear localization of the construct with enrichment at the nuclear periphery. The quantification of Repo-Man distribution was measured as line profile across the nucleus (yellow line) for the experiments in c and g. (e) Profiles of Repo-Man (green) and H3K9me2 (gray) signals across the nucleus. Repo-Man enrichment was measured as the ratio between the average of the two maximum intensity values (Max1 and Max2) by the median of the values in the plateau (c,g). (f) HeLa cells expressing GFP:Repo-Man^N were transfected with control or Nup153 siRNA oligos and the GFP profiles were analysed as in e. Lower panels are representations of GFP:Repo-Man^N localization in a section of a nucleus. (g) Quantification of Repo-Man enrichment at the nuclear periphery in HeLa cells stably expressing GFP:Repo-Man^N or transiently transfected with GFP: Repo-Man^FL (full-length Repo-Man) after RNAi with Control oligos (grey bars) or with a single (Nup153²) or combination (Nup153^1&2) Nup153 oligos (green bars). Data in c and g were analysed with Mann–Whitney test (****P<0.0001), n are depicted in the figures. In box plots in g, central line represents the median, box limits are the 25th and 75th percentiles and whiskers extend to 1.5 × interquartile range.