Figure 7.

The absence of Cdk1 activity does not prevent bipolar spindle formation and metaphase chromosome alignment on release from STLC. (A) After 16-h STLC block and mitotic shake-off, cells were released from STLC by three washes in drug-free medium and were treated with MG132 for 2 h followed by 2-h coincubation with both roscovitine and MG132. Samples were analyzed by 2D FACScan as in Fig 1. Controls were released from STLC for 4 h with no further drug treatment. Percentages shown indicate the subpopulations that were positive for MPM-2. (B) Confocal microscopy was performed on identically treated cells, which were stained for tubulin (green) and DNA with propidium iodide (PI; red). The combined FACScan and microscopy data indicate that controls released from STLC form normal bipolar spindles and exit normally from mitosis. In contrast, cells released from STLC in MG132 remain mitotic but within 2 h have normal mitotic spindles with chromosomes aligned to a normal metaphase plate. After an additional 2 h without Cdk1 activity (MG132 plus roscovitine), cells remained mitotic with normal bipolar spindles and metaphase chromosome alignment. Bar, 16.5 μm.