Figure 3.

Loss of Cdk1 activity in the absence of proteasome activity does not lead to mitotic exit. (A) After STLC treatment and mitotic shake-off, cells were treated with roscovitine in the absence or presence of MG132 for 2 h. Fixed cells were then stained for immunofluorescence microscopy with antitubulin, phosphorylated histone H3 ((PS10)H3), MPM-2, or anti–lamin B antibodies (green) and were counterstained with propidium iodide (PI; red). Roscovitine induced rapid mitotic exit, as evidenced by loss of the monoastral spindles, (PS10)H3, MPM-2 staining, and the assembly of nuclear lamina surrounding decondensed chromatin. However, cells treated with STLC plus roscovitine and MG132 retained monoastral spindles, (PS10)H3, and MPM-2 staining, whereas nuclear lamina were absent. These results were parallel to control cells blocked in mitosis with STLC. Microscope settings were held constant for all image acquisitions. (B and C) The percentage of cells with monoastral spindles (B) or positive for (PS10)H3 (C) were quantitated. The data represent the mean of three counts of >60 cells per count. Gray bar, cells at time 0, the time of mitotic cell selection by shake-off; white bars, cells treated with roscovitine alone; black bars, cells treated with roscovitine plus MG132. In all cases, cells were in the continuous presence of STLC. Error bars represent SD. Bar, 16.5 μm.