Repo-Man and Sds22 are required for timely dephosphorylation of the chromatin-targeted FRET biosensor. (A) Confocal time-lapse images of HeLa Kyoto cells expressing the H2B-targeted Aurora B FRET biosensor, 42 h after transfection of a nontargeting control siRNA (siControl), or siRNAs targeting Repo-Man or Sds22. YPet/CyPet emission ratios are displayed in pseudocolor. Time is shown in min/s. Bars, 10 µm. (B–F) Quantification of YPet/CyPet emission ratios from cells, as shown in A, normalized (norm.) to mean YPet/CyPet ratios of control RNAi cells in metaphase. Curves and bars display mean ± SEM. Videos were aligned to the onset of chromosome segregation (t = 0 min; dashed lines). (B and C) Phosphorylation of the chromatin-targeted biosensor after transfection of different siRNAs targeting Repo-Man or Sds22. n ≥ 12 cells. (D and E) Phosphorylation of the chromatin-targeted biosensor after transfection of siRNAs targeting Mklp2, Repo-Man, or Sds22. n ≥ 10 cells. (F) Phosphorylation of the chromatin-targeted biosensor after transfection of siRNAs targeting Repo-Man or Sds22. ZM1 was added to a final concentration of 2 µM immediately after the onset of chromosome segregation. n ≥ 14 cells. (G) Phosphorylation kinetics of a cytoplasmic Aurora B biosensor (Fuller et al., 2008). Normalized YPet/CyPet emission ratios of live HeLa cells stably coexpressing the cytoplasmic biosensor and H2B-mCherry. Mean ± SEM. n ≥ 18 cells. t = 0 min at chromosome segregation onset (dashed line).