Fig. 3: BRISC deficiency leads to Aurora B hyperactivation and erroneous kinetochore–microtubule (KT-MT) attachments.

a Representative confocal images showing Aurora B activation in Abro1-knockout (Abro1-KO) or BRCC36-knockout (BRCC36-KO) HeLa cells. Cells were treated with 30 ng/mL nocodazole for 4 h and released into fresh medium for 30 min, fixed and stained with anti-Aurora B-pT232 (green) and anti-Aurora B (red) antibodies. DNA was stained with DAPI (blue). Bar, 5 μm. b Box-and-whiskers plots showing the quantification of the relative intensity of Aurora B-pT232 as compared with Aurora B, shown in a. Boxes show the upper and lower quartiles with a line at the median. Whiskers as the extent of 100% of the data. ***p < 0.001 versus CT, one-way ANOVA test, calculated with ZEN3.1 and GraphPad Prism 8 (n = 15 cells and 6 areas for each cell were counted). c, e Representative confocal images showing Aurora B catalytic activity on its substrates, Hec1 and MCAK, in Abro1-KO or BRCC36-KO HeLa cells. Cells were treated as described in a and stained with the indicated antibodies. Hec1 (green)；Hec1-pS55 (red)；MCAK (red); CREST (green); DNA (DAPI, blue). Bar, 5 μm. d Quantification of the relative intensity about Hec1-pS55, as compared with Hec1, shown in c. f Quantification of the relative intensity about MCAK, as normalized to the general kinetochore marker CREST, shown in e. d, f Boxes show the upper and lower quartiles with a line at the median. Whiskers as the extent of 100% of the data. ***p < 0.001 versus CT, one-way ANOVA test, calculated with ZEN3.1 and GraphPad Prism 8 (n = 15 cells and 6 areas for each cell were analyzed). g Representative confocal images showing abnormal KT-MT attachments in Abro1-KO or BRCC36-KO cells. HeLa cells were treated with double thymidine block, then released for 6 h, followed by 10 μM MG132 treatment for 4 h to prevent cells from metaphase to anaphase transition. The cells were fixed on ice to destabilize non-kinetochore microtubules and stained for CREST (green) and α-tubulin (red). Bar, 5 μm. h The percentage of paired kinetochores not attached to microtubules in all counted KT-MT connections. i The percentage of misshapen (aggregated or rod-shaped) kinetochores in all counted paired kinetochores. h, i Data are shown as means ± SD, n = 3 independent experiments. In each independent experiment, 30 cells, five paired kinetochores per cell were counted. **p < 0.01 versus CT, Student’s t-test. j Quantification of the interkinetochore distances shown in Supplementary Fig. 3a. n = 20 cells and five paired kinetochores for each cell were counted. **p < 0.01 versus CT, One-way ordinary ANOVA test. k Representative confocal images showing lateral KT-MT attachment in Abro1-KO or BRCC36-KO cells. HeLa cells were treated as described in g. α-tubulin, red; Astrin, green；CREST, purple; and DNA (DAPI, blue). Bar, 5 μm. l The percentage of cells showing Astrin lateral attachments. Data are shown as means ± SD, n = 3 biological replicates. In each independent experiment, 15 cells were analyzed. **p < 0.01, ***p < 0.001 versus CT, one-way ANOVA test, calculated with GraphPad Prism 8.