Haspin inhibitors reduce H3T3ph and displace Aurora B from centromeres but not the central spindle. (A) HeLa cells were released from double thymidine block and, after 7 h, 5 µM nocodazole was added for 6 h. Mitotic cells collected by “shake-off” were replated in 5 µM nocodazole, 20 µM MG132, and Haspin inhibitors for 1 h. Immunoblots of cell lysates are shown. (B) U2OS cells were released from thymidine block and, after 7 h, 33 nM nocodazole was added. After another 4 h, 0.33 µM nocodazole, 20 µM MG132, and kinase inhibitors were added for 1 h before fixation and immunofluorescence microscopy. To visualize residual H3T3ph, two different red channel exposure times are shown. (C) The ratio of centromere to chromosome arm Aurora B intensity was determined for cells treated as in B (15 centromeres/cell; n = 8 or 9 cells). Means + SD are shown (error bars); ***, P < 0.001 vs. DMSO. (D) Asynchronous U2OS cells were treated with Haspin inhibitors for 2 h. Immunofluorescence microscopy of anaphase cells is shown. Bars, 5 µm.