Fig 2: H3K27ac is a reliable marker of micronuclei membrane integrity.(A) Image of an RPE-1 cell containing one intact (top; GFP-NLS+ and H3K27ac+) and one ruptured (bottom; GFP-NLS- and H3K27ac-) MN (outlined). Cells expressed GFP-NLS (green) and were labeled with H3K27ac (red), and DAPI (blue). (B) Quantification of GFP-NLS and H3K27Ac colocalization in MN. 94.8% of GFP-NLS+ MN were H3K27Ac+; 7.7% of GFP-NLS- MN were H3K27Ac+. Barnard’s exact test = P-value < 0.0001. f correlation coefficient = 0.87. N = 3, n = (193, 116). (C) Image of an RPE-1 cell containing one intact (top; GFP-NES-, H3K27ac+) and one ruptured (bottom; GFP-NES+, H3K27ac-) MN (outlined). (D) Quantification of GFP-NES and H3K27Ac colocalization in MN. 98.2% of GFP-NES- MN were H3K27ac+; 13.1% of GFP-NES+ MN were H3K27ac+. N = 3; n = (117, 137); Barnard’s exact test = P-value < 0.0001. f correlation coefficient = -0.85. (E) Method for inducing MN in RPE-1 cells and fixation timepoints 20, 24, or 30 h after Cdk4/6i release into Mps1i. (F) Quantification of MN rupture frequency, as determined by H3K27ac signal. 52.1% of MN are intact 24 h post Cdk4/6i release. Chi-square = P-value > 0.0001. N = 3, n = (312, 334, 322). (G) Example images of intact single chromosome MN (outlined), defined as H3K27ac+ with a single CENPA focus, and corresponding nuclei in RPE-1 cells 24 h post-Cdk4/6i release. HSA-1, -11, -15, -17, and -20 were probed using centromere specific FISH probes. HSA-4, -13, -18, -19, and -22 were probed using whole-chromosome FISH probes. (H) Chromosome features: Karyotype refers to chromosome length and centromere positioning. Chromosome length (Mb, megabases) corresponds to human genome assembly GRCh38.p13. Gene density from Worrall et al (2018). Centromere length from human centromere assembly GCA_000442335.2 (Miga et al, 2014). (I) Dual parameter plots of RPE-1 cells labeled with DAPI (Pacific-Blue) and EdU (PE) 20, 24, and 30 h post-Cdk4/6i release into Mps1i. (J) Mean proportion of RPE-1 cells in G1, S, and G2 phase by dual label FACS analysis. N = 3, n > 6,000 cells per replicate. All scale bars in figure = 10 µm.

Fig 3: Deletion of HJURP impaired multiple myeloma cell growth and induced apoptosis. A, Expression levels of HJURP protein and mRNA were detected upon HJURP shRNA transfection or the control vector in KMS28BM and H929 cells. B, Proliferation of KMS28BM and H929 cells was significantly inhibited upon HJURP silencing. The cell proliferation was tested every 24 hours using CTG assays. C, Immunoblot analysis for apoptotic markers (PARP and cleaved-PARP) in CTRL or shHJURP–transfected KMS28BM and H929 cells. The immunoblot results of HJURP-knockdown in H929 cells were based on the same batch of H929 cell lysates performed at the same time, and the same GAPDH blot was used as a loading control in A and C. D, KMS28BM and H929 cells were transfected with the CTRL or HJURP shRNA vector for 72 hours. Examination for apoptotic was performed by Annexin V staining and flow cytometry. The percentage of apoptotic cells is represented in a bar diagram from three independent experiments. E, Representative images of metaphase and anaphase of scramble and shHJURP#2 of H929 cell line. Empty arrowheads, lagging chromosomes; arrowheads, chromosomal bridges; arrows, misaligned metaphase. Scale bars, 10 µm. F and G, Frequency of anaphase defects, including lagging chromatin, anaphase bridges, or both (F) and metaphase defects (e.g., misalignment; G) in shHJURP#2 H929 versus scramble. H, DAPI (blue) and CENP-A (green) staining in scramble and shHJURP#2 H929 cell line. Quantification was performed in duplicates (n = 100 per sample); data are mean ± SEM. Scale bars, 10 µm. I, Expression of HJURP protein and mRNA were detected upon HJURP shRNA transfection or cotransfected with HJURP shRNA plus HJURP overexpression vector in H929 cell. GAPDH was used as a loading control. J, Cell viability was measured in shHJURP–transfected H929 cells, shHJURP–transfected H929 cells stably overexpressing HJURP, or a control shRNA. For each condition, cell viability was measured every 24 hours and compared with day 1. K, The percentage of apoptotic cells was measured in shHJURP–transfected H929 cells, shHJURP–transfected H929 cells stably overexpressing HJURP, or a control shRNA. Data are presented as mean ± SD of six replicates out of the representative of three independent experiments. Student t tests were performed compared with the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Synergistic inhibition of Aurora-A and Haspin disrupts the mitotic centromere aggregation of Aurora-B and MCAK. A. The kinase activity of Aurora-A and Aurora-B was examined using Western blotting assays after releasing MDA-MB-231 cells from G2/M synchronization to indicated drugs for 30 min. The concentrations of MLN8237 and CHR-6494 were 200 nmol/L. The repeated-measures ANOVA, followed by the least significant difference test was used to evaluate the difference between groups. n = 3 independent experiments; ns, not significant; *P < 0.05; **P < 0.01. B. Left panel, immunofluorescence shows the location of Aurora-B and CENPA. Right panel, the fluorescence intensity of Aurora-B and CENPA was measured. C. The localization of Aurora-B at centromere was analyzed (n = 3 independent experiments; = 60 cells per condition). D. Immunofluorescence shows the location of MCAK. The arrows represent the direction of the line scan of fluorescence intensity for the entire cell. E. The localization of MCAK at the centromere was analyzed. “Strong” means that more than 50% of the centromeres show MCAK localization, while “weak” indicates that less than 50% of the centromeres show MCAK localization (n = 3 independent experiments; = 123 cells per condition) Abbreviations: MLN, MLN8237; CHR, CHR-6494; DAPI, 4', 6-diamidino-2-phenylindole

Fig 5: Properties of the CENP-LN vault(A) The isolated CENP-LN vault with highlighted CENP-N pyrin domain (∼80 residues, greencyan), CENP-N LNHD (LN homology domain), dimerization domains of CENP-N and CENP-L, and CENP-L LNHD. A 2-fold pseudosymmetry axis (interrupted line with red oval) relates the LNHDs.(B) Top: Rotated cartoon model of the CENP-N-terminal region bound to a CENP-A nucleosome (PDB: 6C0W) in two orthogonal orientations (Pentakota et al., 2017). The pyrin domain binds the L1 loop of CENP-A (light blue) and the LNHD to DNA. H2A C-terminal tails were removed for clarity. Bottom: Superposition of CENP-N in 6C0W to CENP-N in human CCAN predicts a steric clash of the second DNA gyre (boxed) onto pillar 2. The vault can only accept one gyre of DNA.(C) Cartoon model of the RdgC homodimer highlighting the LNHDs and other structural elements.(D) The LNHDs of CENP-LN displayed in absence of other domains (dimerization, pyrin) and modeled with 15-bp DNA from the 6C0W structure (B) positioned identically relative to CENP-N. The 2-fold pseudosymmetry axis between LNHDs coincides with the 2-fold pseudosymmetry axis of DNA.(E) A 90°-rotated view of the same object.