CDDO-Im + bortezomib alters mitochondrial membrane potential (ΔΨm), generation of superoxide (O₂^-), release of mitochondrial proteins cytochrome c and Smac, and activation of caspases. (A) MM.1S cells were treated with CDDO-Im (80 nM), bortezomib (2 nM), or CDDO-Im + bortezomib for 12 hours; incubated with CMXRos for the last 20 minutes; and analyzed by flow cytometry to assay for alterations in ΔΨm. Increase in the number of CMXRos-negative cells indicates loss in ΔΨm. Results are mean ± SD of 3 independent experiments (P < .003). (B) MM.1S cells were treated with CDDO-Im (80 nM), bortezomib (2 nM), or CDDO-Im + bortezomib for 12 hours; harvested; stained with membrane-permeable dye dihydroethidium (HE) for the last 15 minutes; and analyzed by flow cytometry. Results are mean ± SD of 3 independent experiments (P < .005). Superoxide anions oxidize HE to fluorescent ethidium, permitting analysis by flow cytometry. (C) MM.1S cells were treated with CDDO-Im (80 nM), bortezomib (2 nM), or CDDO-Im + bortezomib for 24 hours and harvested; cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti-Smac (top blot) or anti–cyto-c (middle blot) Abs. As a control for equal loading of proteins, filters were also reprobed with antitubulin Ab (bottom blot). Densitometric analysis of the immunoblot demonstrated that CDDO-Im + bortezomib induces a 6- to 7-fold increase in cytosolic cyto-c and Smac levels compared with untreated cells. Blots are representative of 3 independent experiments. (D) MM.1S cells were treated with CDDO-Im (80 nM), bortezomib (2 nM), or CDDO-Im + bortezomib for 24 hours and harvested; cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anticaspase-9 Abs. Blots are representative of 3 independent experiments. FL indicates full length, and CF denotes cleaved fragment. (E) MM.1S cells were treated with CDDO-Im (80 nM), bortezomib (2 nM), or CDDO-Im + bortezomib for 24 hours; cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anticaspase-8 and anticaspase-3 Abs. Blots are representative of 3 independent experiments. (F) MM.1S cells were treated with CDDO-Im + bortezomib alone (□) or in the presence of caspase-8 inhibitor (○), caspase-9 inhibitor (▪), or pancaspase inhibitor () for 24 hours and harvested and assessed for viability using MTT assays. Results are mean ± SD of 3 independent experiments (P < .005).