Fig 1: Pools of Aurora B and Bub1 are remotely regulated from the centromere/kinetochore in a kinetochore distance-dependent manner. (A–D) The kinetochore localization of Mad2 decreases in a kinetochore-microtubule attachment-dependent manner. (A) Kinetochore distances were expanded by CAPH RNAi treatment, and shortened by taxol treatment. Time schedules are shown in Fig. S2C, and a schematic diagram is shown in Fig. S2D. HeLa cells treated with siCAPH or siLuciferase (control) were released from RO3306 block and then treated with colcemid (prometaphase) or MG132 (metaphase). After treatment with or without taxol for 30 min, cells were fixed and stained with anti-Mad2 and Cenp-C antibodies. The following 5 conditions with altered kinetochore-distances were prepared as shown in Fig. S2C and D: colcemid treatment (prometaphase), MG132 treatment (metaphase), MG132 + CAPH RNAi treatment (metaphase with expanded kinetochore-distances), MG132 + taxol (metaphase with shortened kinetochore-distances) and MG132 + CAPH RNAi + taxol (kinetochore-distances once expanded by CAPH RNAi treatment were then shortened by taxol treatment). Scale bar, 5 μm. (B) The inter-kinetochore distance between paired Cenp-C signals of (A) were measured. Bars represent means. Dot plot (N = 60 kinetochore pairs, 3 independent experiment, 5 cells per experiment, 4 kinetochore pairs per cell). (C) Relative intensities (Mad2/Cenp-C) of (A) are shown. Bars represent means. Dot plot (N = 60 kinetochore pairs, 3 independent experiment, 5 cells per experiment, 4 kinetochore pairs per cell, Mann–Whitney U-test, n.s. not significant, ***p < 0.001). (D) Relative intensities (Mad2/Cenp-C) of (A) are shown versus the distance between kinetochores. Scatter plot. (E–I) The kinetochore localization of Bub1 decreases in a kinetochore distance-dependent manner. (E) HeLa cells synchronized and treated as in (A) were stained with Bub1, HH2A pT120 and Cenp-C antibodies. Scale bar, 5 μm. (F, H) Relative intensities (Bub1/Cenp-C or HH2A pT120/Cenp-C) of (E) are shown. Bars represent means. Dot plots (N = 60 kinetochore pairs, 3 independent experiment, 5 cells per experiment, 4 kinetochore pairs per cell, Mann–Whitney U-test, n.s. not significant, ***p < 0.001). (G, I) Relative intensities (Bub1/Cenp-C or HH2A pT120/Cenp-C) of (E) are shown versus the distance between kinetochore pairs. Scatter plots. (J–N) The centromeric localization of Aurora B decreases in a kinetochore distance-dependent manner. (J) HeLa cells synchronized and treated as in (A) were stained with Aurora B, pAurora B and Cenp-C antibodies. Scale bar, 5 μm. (K, M) Relative intensities (Aurora B/Cenp-C or pAurora B/Cenp-C) of (J) are shown. Bars represent means. Dot plots (N = 20 kinetochore pairs, 5 cells per experiment, 4 kinetochore pairs per cell, Mann–Whitney U-test, n.s. not significant, ***p < 0.001). (L, N) Relative intensities (Aurora B/Cenp-C or pAurora B/Cenp-C) of (K) are shown versus the distance between kinetochore pairs. Scatter plots.