Effects of 5-ITu on spindle checkpoint signaling. (A) Asynchronously growing HeLa cells were treated with 3.3 µM nocodazole. After 6 h, mitotic cells were isolated by shake-off, replated, and inhibitors were added at the indicated concentrations under continued presence of nocodazole. Low-resolution live-cell imaging was then started. Cell flattening indicated mitotic exit. At least 50 cells per condition were analyzed. (B) HeLa cells expressing H2B-Cherry were transfected with the CENP-B–INCENP-GFP fusion construct, and 38 h after transfection they were exposed to 330 nM nocodazole for 4.5 h to cause mitotic arrest. Mitotic cells were harvested and nocodazole washed out. After 50 min, cells were either left untreated, or were treated with Reversine or 5-ITu. The timing of anaphase onset was monitored by high-resolution time-lapse video microscopy. The number of analyzed cells is indicated.