Repo-Man and Sds22 function downstream of Aurora B. (A) Confocal time-lapse imaging of HeLa Kyoto cells stably expressing Aurora B–EGFP and H2B-mCherry. Time is shown in min/s. t = 0 min at chromosome segregation onset. Bars, 10 µm. (B) Quantification of Aurora B–EGFP levels on chromatin by ratios of mean Aurora B–EGFP fluorescence divided by mean H2B-mCherry fluorescence. t = 0 min at the onset of chromosome segregation (dashed line). Fluorescence ratios of individual cells were normalized to metaphase values. n ≥ 18 cells. (C) Immunofluorescence staining with antibodies against Aurora B and Aurora B phosphorylated at Thr232 (pT232). DNA was stained with Hoechst 33342. Representative example images of prometaphase, metaphase, and anaphase cells 42 h after transfection with control, Repo-Man, or Sds22 RNAi are shown. Bar, 10 µm. (D and E) Quantification of Thr232-phosphorylated Aurora B (D) or total Aurora B (E) on chromatin in immunofluorescence images, as shown in C. Fluorescence intensities were normalized to control siRNA–transfected prometaphase cells. The dashed lines separate measurements of phospho–Aurora B levels on chromatin for the different siRNA conditions (left) from ZM1 treatment (inhibitor of Aurora B kinase, used as a positive control). Mean ± SEM. n = 7 (D) or 4 (E) independent experiments (>120 [D] or >70 [E] cells per experimental condition).