Haspin inhibitors compromise the spindle checkpoint response to 5 µM nocodazole. (A) HeLa cells were synchronized by thymidine treatment and, 7 h after release, 5 µM nocodazole was added for 7.5 h. Mitotic cells were harvested by mitotic “shake-off” and replated in the continued presence of 5 µM nocodazole together with 5-iodotubercidin and the Aurora B inhibitor ZM447439. After 13.5 h, total cell lysates were analyzed by immunoblotting. (B) HeLa cells were transfected with EGFP–CENP-B or CENP-B–INCENP–EGFP plasmids between and after double thymidine treatments, and then treated essentially as in A, except that cells were replated on coverslips coated with poly-d-lysine. Mitotic indices were determined from ∼100 cells in each condition by DNA staining and fluorescence microscopy (n = 3). Means + SD are shown (error bars); ***, P < 0.001; ND, not detected. (C and D) As for A, but using LDN-192960 (C) or LDN-211898 (D). Black lines indicate that intervening lanes have been spliced out. (E–H) U2OS cells expressing Histone-H2B-mRFP and γ-tubulin–GFP were synchronized and treated with 5 µM nocodazole essentially as described for HeLa cells in A. Mitotic cells were replated in imaging dishes and kinase inhibitors were added in the continued presence of 5 µM nocodazole. Each symbol represents the time at which a cell began to exit from mitosis or die in mitosis, as determined from live imaging series collected over 15 h.