(A, C) Untreated or siRNA transfected (siBub1, siGl2 for control) HeLa S3 cells were synchronized by thymidine block and released for 10 hr, as indicated (BAY-320 was used at 3 μM, BAY-524 at 7 μM). Cells were fixed and stained for Aurora B, Borealin, INCENP, pS7-CENP-A, pS10-histone H3, MCAK, CREST and DNA (DAPI) and analyzed by IFM. Scale bars represent 10 µm. (B, D) Histograms show quantitative results of the experiments described in (A, C). Measurements represent centromeric levels except for pS10-histone H3 signals, which was monitored along chromosome arms (n = 40–113 cells per condition). Scale bars represent 10 µm, error bars represent SEM. (E) FRET experiments were performed on HeLa Kyoto cells stably expressing chromatin (H2B)- or centromere (CENP-B)-fused FRET reporters for Aurora B activity. Cells were synchronized in mitosis by 6 hr treatment with 3.3 μM nocodazole, before the indicated inhibitors and 20 μM MG132 were added prior to live fluorescence microscopy. Heat-map represents the phosphorylation status of the reporter. Scale bar represents 10 µm. (F) Left panel: scatter plot depicts CFP/FRET emission ratios of reporter targeted to chromatin (H2B; n = 23–52 cells per condition). Right panel: scatter plot depicts TFP/FRET emission ratios of reporter targeted to centromeres (CENP-B, n = 16–34 cells per condition). Bars represent mean values; ***p<0.001 (from unpaired two-tailed Student’s t-test).