MPS1 and CDK regulate CCNB1 kinetochore localization. (A and B) HeLa CCNB1-GFP/MPS1-mCherry (A, left), HeLa CCNB1-GFP (A, right), or hTERT1-RPE1 CCNB1-GFP (B) cells were arrested in mitosis with 0.3 µM nocodazole for 4 h and then treated with 2 µM AZ3146 (+MPS1-i) or 5 µM flavopiridol (+CDK-i) for 10 min. Kinetochores were stained for CREST and BUB1. The numbers inset in the image panels indicate the mean kinetochore signal ± SEM of MPS1, BUB1, and CCNB1 relative to the control (15 kinetochores per cell and 12 cells in each of three independent experiments). (C) Control (siControl) or CCNB1 (siCCNB1) depleted HeLa MPS1-GFP/CCNB1-mCherry cells were imaged every 2 min. Representative images of cells from the point at which cell rounding was first observed, set to 0 min, are shown. (D) Fluorescence intensity (I_t) for total cellular CCNB1-mCherry (CCNB1^total) and MPS1-GFP at kinetochores (MPS1^KT) are plotted over time for single cells (n = 13 for siControl and 22 for siCCNB1). Mean CCNB1-mCherry signal is indicated with a gray dashed line; the light gray area marks the SEM. For MPS1-GFP, color-coded lines show the kinetochore signal from individual cells as a function of time, and the black dots mark the mean intensity. Western blot of the siControl and siCCNB1 cells confirmed depletion of CCNB1; actin was used as a loading control.