Fig. 1: Phosphorylation of Aurora B S227 is required to support the metaphase response to catenation.

a DLD1 cells expressing GFP-Aurora B wild type or S227A were scored for nuclear morphology. The mean ± SD of three independent experiments is graphed (n = ≥200) (one-way ANOVA, *P = ≤0.024; Control 6.76% ± 1.46, WT 11.76% ± 5.63, S227A 27.63% ± 10.07). Scale bar = 5 μm. b Anaphase cells were scored for DAPI-positive bridges or PICH-positive strands. The graph represents the mean ± SD of three independent experiments, >20 cells per condition were scored per experiment (two-tailed Students’ t test, *P = 0.027, DAPI: WT 10.35% ± 7.49, S227A 46.02% ± 25.62, PICH: WT 17.18% ± 14.8, S227A 35.98% ± 15.83). Scale bar = 5 μm. c Representative images of single sister chromatids or retained/catenated sister chromatids after 24 h Shugoshin knockdown (siSgo). Scale bar = 2 μm. d Cells were scored for the percent retained (catenated) sister chromatids. The mean ± SEM of three experiments where ≥15 cells per experiment were analysed is graphed (one-way ANOVA; **P = 0.0061; siControl 47.05% ± 5.75, siTopoIIα 78.84 ± 6.61, WT 46.23 ± 4.13, S227A 83.21 ± 5.561). Scale bar = 5 μm. e DLD1 cells expressing GFP-Aurora B wild type or S227A were treated ± 1 μM ICRF193 immediately prior to time-lapse imaging and scored for time from metaphase to anaphase. Violin plots represent all cells scored, median in red. A minimum of 100 cells per condition in three independent experiments were analysed. (one-way ANOVA; ****P = ≥0.0001). f Immunoblot of DLD1 cells (representative of three independent experiments) enriched in mitosis and treated with ICRF193 (1 µM) ± BLU577 (500 nM). TritonX-100 insoluble fractions were probed for Aurora B phospho-S227, total Aurora B, and TopoIIα. Mean ± SD of the immunoblots is graphed (two-tailed Students’ t test; **P = 0.001; +ICRF/+BLU 0.5835 ± 0.085). g Cells were treated with 500 nM nocodazole and imaged using live-cell time-lapse microscopy for 24 h and scored for their time arrested in mitosis. The mean ± SD of three experiments where >50 cells were analysed per condition, per experiment are graphed. Representative images of (h) BubR1 and (i) MAD2 kinetochore localisation after inducing a metaphase delay with ICRF193 (4 h, 1 µM) ± BLU577 (20 min 500 nM) or ±nocodazole (5 min, 500 nM), from three independent experiments, a minimum of ten images were acquired. DAPI (blue), GFP-Aurora B (green), BubR1/MAD2 (magenta), CREST (white). Scale bar = 5 μm.