Bay 11-7082 markedly enhances UCN-01-induced activation of SAPK/JNK and dephosphorylation of p34^cdc2, whereas enforced MEK/ERK activation fails to block Bay 11-7082/UCN-01-mediated lethality. 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 phosphorylation status of ERK1/2, JNK, c-Jun, p38, and p34^cdc2. For each condition, lanes were loaded with 30 μg protein. Alternatively, U266 cell lysates were subjected to a SAPK/JNK kinase assay. SAPK/JNK activity was reflected by the extent of phosphorylation of a GST-c-Jun fusion protein (A, bottom panel). For panels A and B, results are representative of 3 separate experiments. (C-D) U266 cells were transiently transfected with activated MEK1 (mutation of serine to aspartic acid at sites 218 and 222)/pEGFP-C2 or its corresponding empty vector and isolated using FACS, as described in “Materials and methods.” After 24 hours, purity (C) and viability (greater than 95%) of GFP⁺ cells (gate R2) were monitored by flow cytometry and trypan blue exclusion. GFP⁺ cells were exposed to 200 nM UCN-01 ± 4 μM Bay 11-7082 or 20 μM U0126 for 24 hours, after which the extent of apoptosis was determined by evaluating Wright-Giemsa-stained cytospin slides (D). Values represent means ± SDs for 3 separate experiments performed in triplicate. ^*Significantly lower than values for cells transfected with empty vector (P < .01).