Fig 2: Bub1 regulates CENP-AS7 phosphorylation and Aurora-A targeting at centromeres. a, b HeLa cells depleted of Bub1 by siRNA were immunostained with anti-p-CENP-AS7 (a) or anti-Aurora-A (b) and CREST antibodies. Magnifications (× 9) of single chromosomes are shown. Means ± SEM are shown (on the right of the panel) for the quantification of signal intensity relative to CREST obtained with antibodies against p-CENP-AS7 or Aurora A antibodies in a representative experiment (for p-CENP-AS7 intensity, n = 20 cells in each condition; for Aurora A intensity, n = 25 cells in control and 20 cells in Bub1 RNAi conditions; ****P < 0.0001; two-tailed unpaired nonparametric Mann–Whitney U-test). c Metaphase chromosome spreads from CENP-A-HA-WT, CENP-A-HA-S7A, and CENP-A-HA-S7D cell lines depleted of Bub1 were immunostained with antibodies against Bub1 and Sgo1. Scale bar = 5 µm. Inter-kinetochore distance means ± SEM of five to six representative cells with joined SC are shown (n = 10–50 chromosomes for each cell). Statistical analysis were performed on the mean of the distance measured for each cell (n = 5–6; ****P < 0.0001; ***P < 0.001; NS, not significant; two-way ANOVA with Tukey’s post hoc analysis)

Fig 3: Cellular aberrations in patient fibroblast lines.(A) Immunofluorescence staining of immortalized fibroblasts of index IA; blue—DAPI; magenta—CENPT; green—CENPA or phosphorylated CENPA. Scale bars represent 10µm. Multinucleated cells are depicted in columns 1–3. Micronuclei (solid arrowheads) and chromosome lagging (arrow). (B) Normal mitosis on the left. Examples of multipolar mitosis on the right in immortalized fibroblast of index IA. Blue—DAPI; magenta—CENPT; green–?-tubulin. Scale bars represent 10µm.

Fig 4: CENP-A-S7P is required for the protection of sister chromatid cohesion during mitosis. a After endogenous CENP-A siRNA depletion, metaphase chromosome spreads from CENP-A-HA-WT, CENP-A-HA-S7A, or CENP-A-HA-S7D cell lines were immunostained with an anti-HA antibody. A × 9 magnification of a single chromosome is shown. Scale bar = 5 µm. b Mitotic cells displaying premature sister chromatid separation (PSCS) were quantified with two different clones from each cell line; at least 1000 cells were counted for each data point. The data shown are the mean ± SD (n = 3 experiments; **P<0.01, ***P<0.001, one-way ANOVA)

Fig 5: Aurora A is involved in sister chromatid cohesion. a After transfection with a siRNA against Aurora A or Aurora B, chromosome spreads were prepared from HeLa-S3 cells and immunostained with anti-p-CENP-AS7 and CREST antibodies. A × 9 magnification of a single chromosome is shown. The relative fluorescence intensity of p-CENP-AS7 relative to CREST was determined and mean values ± SEM are shown on the bottom left side of the panel (n = 30 cells for control and Aurora-A RNAi and 20 cells for Aurora-B RNAi, from a representative experiment; ****P < 0.0001; *P < 0.1; NS, not significant; non-parametric Kruskal–Wallis test). Bottom right panel: proportion of mitotic cells with PSCS (n = 4 experiments; > 200 cells per experiment were counted; ****P < 0.0001; ***P < 0.001; NS, not significant; one-way ANOVA) are shown. b HeLa cells were treated with MLN8054 (Aurora A inhibitor). Aurora A inhibition was monitored by western blotting with an anti-p-Aurora-AT288 as a marker of the Aurora-A active form (top of the panel); chromosome spreads were then stained with CREST (red) and anti-p-CENP-AS7 (green) antibodies. A × 9 magnification of a single chromosome is shown. Scale bar = 5 µm. The fluorescence intensity of p-CENP-AS7 signals relative to CREST signals mean values ± SEM are shown on the bottom left side of the panel (n = 25 cells for a representative experiment; ****P < 0.0001; non-parametric Kruskal–Wallis test). The mean ± SD of the percentage of cells displaying PSCS is shown on the bottom right of the panel (n = 3 experiments; > 200 cells per experiment were counted; ***P < 0.001; two-tailed unpaired parametric t test). c Following endogenous CENP-A siRNA depletion, CENP-A-HA-WT, CENP-A-HA-S7A, or CENP-A-HA-S7D cell lines were treated for 1 h with 10 µM MLN8054 Aurora-A-specific inhibitor and then cytospun to determine the proportion of mitotic cell displaying PSCS. The data shown are the mean ± SD (n = 3 experiments; at least 200 cells were counted per condition; **P < 0.01, ***P < 0.001, two-way ANOVA)