Figure 1. Repo-Man is enriched at the periphery of interphase nuclei.

(a) Summary of Repo-Man interactors identified in previous studies⁹,¹¹,¹⁴. In green interactions with nuclear envelope proteins, blue with histones; yellow phosphatases (PP2A—mitotic exit onset only) and (PP1). (b) HeLa cells were transfected with GFP:Repo-Man (red) then fixed and stained for Nup153 (green). (c) HeLa cells immunostained for endogenous Repo-Man (red) (a,b,d,e) transfected with GFP:Nup153 (a,c) or co-immunostained for endogenous Nup153 (green) (d,f). Example of PLA signals (red) using Repo-Man and Nup153 antibodies (g,h). Scale bar, 10 μm. (d) HeLa cells were transfected with GFP:Nup153 (b,d,f) or GFP alone (a,c,e) and PLA (red) was performed using Repo-Man and GFP antibodies (c,d). (e) Quantification and cellular distribution of PLA signals as described in d from two independent experiments (Fisher ****P-value<0.0001). (f) Percentage and cellular distribution of PLA signals in Repo-Man (green) or Control RNAi (grey) (Chi-Square, ****P-value<0.0001). (g) Electron Microscopy image of Repo-Man cell line expressing the peripheral N terminus domain fused to GFP. Immuno-electron microscopy was conducted using an anti-GFP antibody. Black arrow shows accumulation at the edge of the NPC (white arrow) and Red arrows show accumulation on heterochromatin adjacent to the nuclear envelope (see Supplementary Fig. 1), scale bar, 500 nm. (h) Quantification of the experiment in g. Numbers represent the density of labelling in each of the indicated sub-compartments as the number of gold particles μm⁻² (see materials and methods). Total number of gold particles counted was 1,057.