Stable transfection of DN caspase-9 diminishes Bay 11-7082/UCN-01-induced apoptosis in MM cells. U266 cells were stably transfected with a DN caspase-9 (mutation of cysteine to alanine at the active residue 286) or an empty vector (pcDNA3.1), as described in “Materials and methods.” Transfected cells were treated with 4 μM Bay 11-7082 + 200 nM UCN-01 for 24 hours, after which the extent of apoptosis was monitored by annexin V-FITC staining and flow cytometric analysis as described for Figure 1C; results are representative of 3 separate experiments (A). Cells were treated as described for panel A, and the percentage of cells displaying morphologic evidence of apoptosis or loss of mitochondrial membrane potential (Δψ_m) was determined as described in “Materials and methods.” Values represent means ± SD for 3 separate experiments performed in triplicate. ^**Significantly lower than values for cells transfected with empty vector; P < .002 (B). Alternatively, Western blot analysis was used to monitor the expression/cleavage of the indicated caspases and PARP, the release of cytochrome c and Smac/DIABLO into the cytosolic S-100 fraction (C) or the phosphorylation of IκBα, JNK, and p34^cdc2, and the expression of antiapoptotic proteins (D). (C-D) Each lane was loaded with 30 μg protein; blots were subsequently reprobed for actin expression to ensure equivalent loading and transfer of protein. CF indicates cleavage fragment. Results of a representative experiment are shown; an additional study yielded equivalent results.