Bim plays a functional role in the lethality of Chk1/MEK1/2 inhibition toward G₀/G₁–enriched MM cells. (A) H929 cells were exposed to the indicated concentrations of AZD6244 for 24 hours, followed by 300nM AZD7762 for an additional 24 hours, after which time Western blot analysis was performed to monitor the expression of Bim, including phosphorylated (slow migrating) and unphosphorylated (fast migrating) Bim_EL, as well as Bim_L and Bim_S isoforms. (B) Alternatively, cytosolic and mitochondria-enriched (pellet) extracts were subjected to Western blot analysis to monitor the release of mitochondrial proapoptotic proteins (ie, cytochrome c, AIF, and Smac) and translocation of Bax. Parallel blots probed for Cox-2 (cytochrome oxidase subunit 2, a protein of mitochondrial inner membrane) and β-actin are shown to ensure equal loading and transfer for mitochondrial and cytosolic fractions, respectively. (C) Bim shRNA (shBim, clone W12) and scramble control shRNA (shCtrl) U266 cells were cultured in medium containing 0.05% or 10% FBS for 48 hours, followed by coadministration of 1.5μM AZD6244 for 24 hours ± 300nM AZD7762 for an additional 48 hours. Western blot analysis was performed to monitor Bim_EL expression and PARP degradation. CF indicates the cleavage fragment. (D) shBim (clones W12 and W4) and shCtrl U226 cells were treated as described in panel C, after which time the percentage of dead (7-AAD⁺) cells was determined by flow cytometry and was significantly less (**P < .01 and ***P < .002) than values for shCtrl cells treated identically in 0.05% FBS medium. Inset shows Western blots demonstrating knock-down of Bim in shBim cell, compared with shCtrl cells. For panels A through D (inset), each lane was loaded with 20 μg of protein; blots were stripped and reprobed with anti-actin Ab to ensure equal loading and transfer. Two additional studies yielded equivalent results.