Figure 9. FXR1 regulates G1 cell cycle progression.

• A
  HeLa cells were transfected with the import/export reporter plasmid XRGG‐GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 h to induce XRGG‐GFP nuclear import. Following washout, the nuclear export of XRGG‐GFP was analysed by live video spinning disc confocal microscopy. The selected frames of the movies are depicted in Fig EV5C. The percentage of cytoplasmic XRGG‐GFP over time was quantified in Fig EV5D, and quantifications of individual cells from the 20 and 30 min time points are depicted in (A), and 199 cells were analysed (mean ± SD, *P < 0.05; **P < 0.01; N = 3).
• B
  HeLa cells stably expressing GFP‐Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 h and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.
• C, D
  Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p‐Rb-positive cells was quantified in (D), and 2,800 cells were analysed (mean ± SD, **P < 0.01; N = 3).
• E, F
  Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30 min and analysed by immunofluorescence microscopy. The percentage of p‐Rb- and/or EdU‐positive cells was quantified in (E); 2,100 cells were analysed (mean ± SD, *P < 0.05; **P < 0.01; N = 3), and the percentage of p‐Rb- and/or cyclin B‐positive cells was quantified in (F); 3,300 cells were analysed (mean ± SD, *P < 0.05; N = 3).
• G
  A hypothetical model how fragile X‐related proteins spatially regulate nucleoporin condensation. FXR proteins (blue) interact with cytoplasmic soluble Nups (red circles) and dynein (green) and facilitate their localization to the NE during early G1. This function of FXR proteins inhibits formation of aberrant cytoplasmic Nup assemblies, the cytoplasmic nucleoporin granules (CNGs), contributing to the equilibrium of NE‐NPCs and driving the G1‐specific protein export and maintenance of nuclear shape and cell cycle progression. Silencing of FXR proteins, for instance in FXS patients, leads to the accumulation of CNGs, nuclear atypia, protein export defects, and defects in cell cycle progression, which may contribute to the pathology of FXS.

Data information: Scale bars are 5 μm. Statistical significance was assessed by Kruskal–Wallis test with Dunn's correction (A), unpaired two‐tailed Student's t‐test (D) and one‐way ANOVA test with Sidak's correction (E, F).