Haspin inhibitors compromise KT-MT attachment correction. (A) HeLa cells were released from thymidine treatment and, after 7 h, 100 µM monastrol was added for 3 h to accumulate cells in mitosis with incorrect KT-MT attachments. Then 50 nM Hesperadin was added to inhibit Aurora B, together with 20 µM MG132. After 1.5 h, monastrol and Hesperadin were removed by washing into fresh medium containing Haspin inhibitors or controls in the continued presence of MG132. Approximately 200 cells were classified in each condition by fluorescence microscopy. Means ± SD are shown (error bars), n = 3. (B) Immunofluorescence microscopy of Dsn1-S109ph during Aurora B reactivation in the presence or absence of Haspin inhibitors in cells treated as in Fig. 4 A. (C) Approximately 100 mitotic cells in each condition from one experiment as in B were classified according to phosphorylated Dsn1 (Dsn1-S109ph) or total Dsn1 (Dsn1) staining intensity at kinetochores. Similar results were obtained in a duplicate experiment. (D) HeLa cells were transfected with plasmids encoding CENP-B–EGFP or EGFP–CENP-B–INCENP between and after double thymidine treatments, then treated as in B. (E) The intensity of Dsn1-S109ph staining in EGFP-positive cells from D was classified in one experiment as in C. Similar results were obtained in a duplicate experiment. Bars, 5 µm.