Extended Data Figure 9. Requirements for nucleocytoplasmic compartmentalization.

A. Quantification of NE-sealing from siRNA treated cells as in Figure 4B, (Ctrl 140 cells from 7 independent experiments; UFD1-1, 60 cells from 3 independent experiments, P = 0.044; UFD1-3, 60 cells from 3 independent experiments, P = 0.021; CHMP2B 40 cells from 2 independent experiments, N.S. All times quoted ± S.E.M. in minutes; 2-tailed student’s T-test was used to assess significance at the 85-minute timepoint). B. Resolved cell lysates from A were analysed by western-blotting with the indicated antisera. C. Nuclear envelope integrity assay as performed with cells stably expressing mCh-H2B and GFP-NLS and transfected with the indicated siRNA. Differences in nucleo-cytoplasmic partitioning was assessed after plateau at the 65 minute timepoint using a 2-tailed Student’s T-test : (Ctrl, 79 cells from 4 independent experiments, CHMP2A-1, 60 cells from 3 independent experiments, P = 0.048; CHMP2A-2, 52 cells from 3 independent experiments, P = 0.011; CHMP3, 28 cells from 3 independent experiments, P = 0.028, error bars represent S.E.M.). D, E. HeLa cells stably expressing mCh-H2B and GFP-NLS were transfected with the indicated siRNA and imaged live. 60 minutes post anaphase onset, cytoplasmic signal was photo-ablated (T = 0) and Recovery of cytoplasmic signal from the nuclear pool was calculated for the indicated conditions (Cytoplasmic : Nuclear ratio of GFP-NLS was normalized to T = 0. Ctrl, 21 cells from 4 independent experiments; CHMP2A-1, 24 cells from 4 independent experiments, P = 0.04; CHMP2A-2, 23 cells from 4 independent experiments, P = 0.05; CHMP3, 15 cells from 3 independent experiments, P = 0.004, 2-tailed Student’s t-test was used to assess significance after 10 minutes. In D, error bars represent S.E.M. in E, scale bar is 10 μm. F. Scoring of multinucleate and midbody-connected HeLa cells transfected with the indicated siRNA and stained with anti-tubulin and DAPI (300 cells analysed per condition, n = 4 ± S.D.).