Figure 2.

SP600125 abrogates spindle assembly checkpoint function in human cells. (A) Nocodazole (Noc)-arrested mitotic U2OS cells were collected by shake-off and reseeded into fresh medium containing Noc alone or in combination with the specified drugs. At the indicated time points, cells were collected and analysed for phospho-histone H3 (p-histone H3) positivity. The Histogram shows the relative mitotic index at 3 h after re-seeding in the presence of different drug combinations. For comparison, a fraction of the cells was released from the Noc block and analysed in parallel (release). (B,C) U2OS cells were treated as in (A), using either Noc or taxol, to obtain mitotic cells. After mitotic shake-off, cells were re-seeded into Noc or taxol. (B) Cyclin B-associated kinase activity and cyclin B protein levels were determined at the indicated time points in the specified drug combination. Cdk4 was used as a loading control. (C) Mitotic index, as determined by p-histone H3 positivity at different time points after re-seeding in the specified drug combination. (D) Cells were treated as in (B) and p-histone H3 positivity (bars) and cyclin B-associated kinase activity were determined after 3 h of co-incubation with the indicated SP600125 concentrations or an equivalent amount of solvent (mock).