Journal: bioRxiv
Article Title: RCC1 depletion drives protein transport defects and rupture in micronuclei
doi: 10.1101/2024.09.04.611299
Figure Lengend Snippet: A. Quantification of NUP density in intact RPE1 cell MN. Wilcoxon rank sum test, N=(3), n=(84,76,84,69). One sample Wilcoxon rank sum comparing MN:Nuc to 1, Nup133 p<0.0001, Nup153 p<0.0001, TPR p<0.0001, CRM1 p<0.0001. B. Maximum projections of the top surface of MN in RPE1 cells stained for Nup133, Nup153, TPR, and CRM1. Scale bar =1um. C. Maximum projections of Ran in RPE1 cells 24hrs post BAY addition. Quantification of Ran intensity for nuclei and intact MN. Wilcoxon rank sum test, N=3, n=(88, 140). D. Maximum projections of RCC1 in RPE1 cells 24hrs post BAY addition. Intact MN indicated with white circles. Quantification of RCC1 intensity for nuclei and intact MN. Wilcoxon rank sum test, N=3, n= (168, 268). E. Maximum projected images of RCC1 staining in U2OS cells. Intact MN indicated by white circles. Scale bar= 10um. Quantification of RCC1 intensity for nuclei and intact MN. Wilcoxon rank sum test, N=3, n=(111, 176). F. Maximum projections for RCC1 staining in control and RCC1-H2A expressing RPE1 cells, with examples of low and high expressing cells, 24hrs post BAY. Control and low RCC1-H2A cells are also shown with increased contrast so RCC1 is visible in MN. Scale bar= 5um. G. Quantification of RCC1 intensity in MN, normalized to median RCC1 intensity for each replicate, in control and RCC1-H2A expressing RPE1 cells. RCC1-H2A cells were categorized as high or low based on nuclear RCC1 levels. H. MN:Nuc ratio of NES-GFP-NLS intensity in MN for control and RCC1-H2A expressing cells. G,H: Wilcoxon rank sum test, N=4, n=(270, 149, 36). I. MN stability in RPE1 cells for control and RCC1-H2A expression. Barnards test. N=3, n=(294, 349, 73). For all graphs, ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Article Snippet: Primary antibodies used: mouse anti-H3K27ac (1:250; 1 h; 39085, Active Motif) rabbit anti-H3K27Ac (1:500; ab4729; Abcam), rabbit anti-H3K27me3 (1:500; MA511198; Thermo Fisher), rabbit anti-H3K9me2 (1:250; AM39041; Active Motif), rabbit anti-Phospho-Rpb1 CTD (Ser2) (1:500; 13499; Cell Signaling Technology), human anti-CREST (1:100; 1 h; 15-234; Antibodies Incorporated), rabbit anti-LBR (1:500; ab32535; Abcam), rabbit anti-Lamin A (1:500, O/N at 4°C; L1293; Sigma Aldrich), mouse anti-Ran (1:500; O/N at 4°C; MA511198; Thermo Fisher), mouse anti-RCC1 (1:200; 2 h, refix with 4% pfa; sc-376049; Santa Cruz Biotechnology), rabbit anti-Nup133 (1:100; ab155990; Abcam), rabbit anti-Nup153 (1:100; ab96462; Abcam), rabbit anti-TPR (1:500; ab84516; Abcam), rabbit anti-CRM1 (1:1000; 46249; Cell Signaling Technology), RFP-boost (1:1000; 1 h; rb2AF568; Chromotek), rabbit anti-FLAG (1:500; F7425; Millipore Sigma).
Techniques: Staining, Control, Expressing
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