EB1 and microtubules are required for Aurora B to phosphorylate kinetochores and chromatin substrates in prometaphase. (A) Experimental outline of B and C. (B) HeLa cells treated with ZM, MG132, and monastrol were washed out of ZM to monitor recovery of phospho–histone H3Ser10 (pH3S10) after 15 min. EB1-depleted cells are compared with control cells treated similarly and fixed immediately (ZM washout, 0 min) or replaced in ZM-free media and fixed after 15 min (ZM washout, 15 min). Bar, 1.6 µm. (C) Spreading of Aurora activity from centromeres to kinetochores (pKNL1) requires EB1. Note that centrosomal staining is an artifact, and kinetochore staining represents phospho-KNL1. Bar, 1.7 µm. (D) Quantification of the mean total pH3S10 recovery in B. *, P = 2.2 × 10−5. Error bars show standard deviations. (E) Quantification of kinetochore phosphorylation in C. (F) Centromeric Aurora B levels from B. **, P = 5.3 × 10−157. (G) Microtubules are required for the recovery of inner centromeric Aurora B and KNL1 phosphorylation after ZM washout. Note that the phospho-KNL1 has nonspecific staining of centrosomes (Welburn et al., 2010). Bar, 1.4 μm. (H) Quantification of inner centromeric Aurora B intensities in G (n > 160). Recov, recover; Noc, nocodazole. *, P = 4.19 × 10−91. (Scheme for the experiment is shown in Fig. S5 B.) The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. a.u., arbitrary units.
