Coadministration of Bay 11-7082 and UCN-01 activates the mitochondrial pathway and caspase cascade in association with interruption of the NF-κB pathway. MM cells were exposed to 200 nM UCN-01 ± 4 μM (A; U266) or 1 μM (B; MM.1S) Bay 11-7082 for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor the expression of caspase-9 and -8 and PARP. Alternatively, cytosolic fractions were prepared as described in “Materials and methods,” and expression of cytochrome c and Smac/DIABLO in cytosol was assessed by Western blot analysis (U266 cells) (A). For each condition, lanes were loaded with 30 μg protein; blots were subsequently reprobed for actin expression to ensure equivalent loading and transfer of protein. CF indicates cleavage fragment. (C) U266 cells were treated as described for panel A, after which nuclear extracts were prepared and subjected to EMSA, as described in “Materials and methods.” The activity of NF-κB was reflected by the extent of binding of ³²P-labeled oligonucleotides corresponding to the NF-κB binding site of the immunoglobulin light chain promoter. To control for probe specificity, nuclear extracts obtained from untreated cells were incubated with a 100-fold excess of unlabeled NF-κB oligonucleotides for 10 minutes before the addition of labeled NF-κB oligonucleotides (C, lane 1; Ctrl+C'). For panels A-C, the results of a representative experiment are shown; 2 additional studies yielded equivalent results. (D) Both U266 and MM.1S were exposed to 200 nM UCN-01 with or without 50 μg/mL SN50 (specific inhibitory peptides of NF-κB) or a nonfunctional mutant control (SN50M) for 24 hours, after which the extent of apoptosis was determined by evaluating Wright-Giemsa-stained cytospin slides; values represent means ± SDs for 3 separate experiments performed in triplicate).