PD184352 blocks UCN-01–mediated Bim_EL phosphorylation and down-regulation in human MM cells. (A) U266 cells were exposed to 100 nM UCN-01 ± 5 μM PD184352, and at the indicated intervals the cells were harvested and subjected to Western blot analysis to monitor phosphorylation of ERK, expression of Bim, as well as cleavage of caspase-9 and -3. (B) RPMI8226 cells were treated with 150 nM UCN-01 plus or minus 5 μM PD184352 for 24 hours, after which Western blot analysis was performed to assess ERK activation, Bim_EL phosphorylation, and cleavage of caspase-3 and PARP. (C) U266 and RPMI8226 were exposed to UCN-01 (U266, 100 nM; RPMI8226, 150 nM) plus or minus PD184352 (5 μM) or PD98059 (50 μM) for 24 hours (RPMI8226) or 40 hours (U266), after which the percentage of apoptotic cells (annexin V⁺) was determined by flow cytometry as described in “Materials and methods.” Results represent the means (± SD) for 3 separate experiments performed in triplicate. **Significantly greater than the value for cells treated with UCN-01 alone (P < .001). (D) U266 and RPMI8226 cells were treated as described for panel C, after which cells were lysed and subjected to Western blot analysis to examine expression of other Bcl-2 family members using the indicated primary antibodies. For panels A, B and D, each lane was loaded with 20 μg protein; the blots were subsequently stripped and reprobed with antitubulin antibody to ensure equal loading and transfer. Phosphorylated forms of Bim_EL are manifested by slowly migrating species (arrows). CF indicates cleavage fragment. Two additional studies yielded equivalent results.