Localization of Aurora B in HCT116 WT and Haspin CM cells with or without Bub1 inhibition. (A) IF images of HCT116 WT or Haspin CM cells ±10 µM BAY-320 and arrested in mitosis using nocodazole (scale bar, 5 µm). (B and C) IF intensity levels of H2AT120ph (B) and Aurora B (C) at centromeres were quantified. Levels were normalized over CENP-C. The graphs show the mean and SD. A minimum of 25 cells was quantified per condition. Data are representative of two independent experiments. P values were calculated using a two-way ANOVA with Tukey’s multiple comparison test. n.s., not significant. (D) IF images of chromosome spreads prepared from HCT116 WT and Haspin CM cells ±10 µM BAY-320 (scale bar, 5 µm). The insets show the magnification of the boxed region (scale bar, 1 µm). (E) CENP-C/CENP-C distances from individual pairs of kinetochores (dots) and the mean (bar) and SD. A minimum of 103 kinetochore pairs was measured per condition. P values were calculated using a one-way ANOVA with Tukey’s multiple comparison test. n.s., not significant. (F) Line plots depicting IF intensity levels of Aurora B and CENP-C, measured along a line that intersects the two sister CENP-C signals of the inter-kinetochore axis (scale bar, 1 µm). The Aurora B levels are normalized to the CENP-C signals. IF images of the specific kinetochore pairs represented in the line plots are shown on the left. The dotted lines in the CENP-C image correspond to the line plots.