Activation of SAPK/JNK plays a functional role in Bay 11-7082/UCN-01-mediated down-regulation of antiapoptotic molecules and apoptosis in MM cells. (A) U266 cells were treated with 200 nM UCN-01 + 4 μM Bay 11-7082 in the presence and absence of the JNK inhibitor SP600125 (20 μM) for 24 hours, after which the percentage of cells displaying morphologic features of apoptosis or loss of mitochondrial membrane potential (Δψ_m) were determined by evaluating Wright-Giemsa-stained cytospin preparations and monitoring DiOC₆ uptake by flow cytometry, as described in “Materials and methods.” Values represent means ± SDs for 3 separate experiments performed in triplicate. ^*Significantly lower than values for cells treated with Bay 11-7082/UCN-01 in the absence of SP600125; P < .01. (B) U266 cells were treated with 200 nM UCN-01 + 4 μM Bay 11-7082 in the presence of a specific JNK inhibitory peptide (D-JNKI1; 1 μM) or its corresponding control peptide (D-TAT, 1 μM) for 24 hours, after which the extent of apoptosis were monitored by annexin V-FITC staining and flow cytometric analysis as described for Figure 1C. Results are representative of 3 separate experiments. (C) U266 cells were treated as in panel A, after which they were lysed and subjected to Western blot analysis to monitor expression of the indicated proteins. Alternatively, cytosolic S-100 fractions were prepared as described in “Materials and methods,” and the expression of cytochrome c and Smac/DIABLO was assessed by Western blot analysis. For each condition, lanes were loaded with 30 μg protein; blots were subsequently reprobed for actin to ensure equivalent loading and transfer of protein. Two additional studies yielded equivalent results. CF indicates cleavage fragment.