Fig. 5
CENP-AS7 phosphorylation is involved in sister chromatid cohesion and Sgo1 maintenance at centromeres in response to tension. a Chromosome spreads from CENP-A-HA-WT, CENP-A-HA-S7A, and CENP-A-HA-S7D cells depleted of endogenous CENP-A were immunostained for Sgo1 and HA. Magnifications (× 9) are shown. In the CENP-A-HA-S7A cell line, the presence and absence of Sgo1 immunostaining at the centromere of joined (empty arrow) or separated sister chromatids (full arrow and asterisk), respectively, are shown. Scale bar = 5 µm. b CENP-A-HA-WT and CENP-A-HA-S7A cell lines were treated or not with nocodazole for 3 hours. The means ± SD of PSCS percentages are shown on the left (n = 2 experiments; > 150 cells per experiment were counted; ****P < 0.0001; ***P < 0.001; NS, not significant; two-way ANOVA). Quantifications of Sgo1 in cell lines presenting joined or separated sister chromatids (SC) are shown on the right. The graph shows the means ± SEM from a representative experiment (n = at least 22 cells; ****P < 0.0001; ***P < 0.001; **P < 0.01; NS, not significant; non-parametric Kruskal–Wallis test). c CENP-A-HA-WT, CENP-A-HA-S7A and CENP-A-HA-S7D cell lines depleted from endogenous CENP-A were treated with 20 µM MG132 for 6 h and PSCS were scored. Means ± SD for the proportion of cells displaying PSCS are shown (n = 4 experiments; > 150 cells per experiment were counted; ****P < 0.0001; ***P < 0.001; **P < 0.01; two-way ANOVA). d Means ± SEM from a representative experiment quantifying Sgo1 fluorescence intensity on joined or separated SC after the depletion of endogenous CENP-A in CENP-A-HA-WT, CENP-A-HA-S7A, and CENP-A-HA-S7D cell lines further treated with 20 µM MG132 for 6 h (n = 23–26 cells, except for S7D with separated SC where n = 14; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.1; NS, not significant; non-parametric Kruskal–Wallis test). e CENP-A-HA-WT and CENP-A-HA-S7A cell lines depleted from endogenous CENP-A and synchronized as indicated were scored for PSCS after 3 or 6 h following nocodazole or MG132 treatment. The means ± SD of PSCS percentages are shown on the left (n = 2 experiments; > 200 cells per experiment). Right panel: mitotic indexes (n = 2 experiments, > 400 cells per experiment)
