FIG. 8.
CDK inhibitors inhibit mitotic Bcl-xL/Bcl-2 phosphorylation, which is normally transient but is sustained if mitosis is prolonged. (A) Inhibition of CDK inhibits mitotic Bcl-xL/Bcl-2 phosphorylation. KB-3 cells were synchronized at the G1/S boundary by a double thymidine block, and at 7 h after release, they were either harvested or treated for 2 h with 25 μM Ros, 1 μM PA, 1 μM RO, or the DMSO vehicle (Veh). Whole-cell extracts were prepared and immunoblotted for the indicated proteins. Arrows indicate phosphorylated (p) forms of Bcl-xL or Bcl-2. GAPDH was used as a loading control. (B) Prolongation of mitosis without microtubule inhibition leads to sustained Bcl-xL/Bcl-2 phosphorylation. KB-3 cells were synchronized at the G1/S boundary by a double thymidine block and were released into complete medium. Two flasks of cells were left untreated and were harvested at 1 h and 10 h postrelease as controls. Other flasks of cells were treated with the DMSO vehicle or with MG132 (20 μM) at 10 h postrelease and were harvested at the indicated time points following release. Whole-cell extracts were prepared and immunoblotted for the indicated proteins. One flask of synchronized cells was treated with 30 nM vinblastine (VBL) for 24 h as a positive control for sustained mitotic arrest. Arrows indicate phosphorylated (p) forms of Bcl-xL or Bcl-2. GAPDH was used as a loading control.