PK + bortezomib changes mitochondrial membrane potential (Δψm), generation of superoxide (⁠

⁠), release of mitochondrial proteins Cyto-c and Smac, and activation of caspase-9. (A) MM.1S cells were treated with PK (50 μM), bortezomib (2 nM), or PK + 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 means ± SDs of 3 independent experiments (P < .005). (B) MM.1S cells were treated with PK (50 μM), bortezomib (2 nM), or PK + bortezomib for 12 hours; harvested; stained with membrane permeable dye dihydroethidium (HE) for the last 15 minutes; and analyzed by flow cytometry. Results are means ± SDs 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 PK (50 μM), bortezomib (2 nM), or PK + bortezomib for 24 hours and harvested; cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti–cyto-c (top panel) or anti-Smac (middle panel) Abs. As a control for equal loading of proteins, filters were also reprobed with antitubulin Ab (bottom panel). Blots are representative of 3 independent experiments. IB indicates immunoblot. (D) MM.1S cells were treated with PK (50 μM), bortezomib (2 nM), or PK + bortezomib for 24 hours. Cytosolic extracts were assayed for protease activity using LEHD-pNA as substrate as per the manufacturer's instructions (colorimetric assay kit; Biovision, Palo Alto, CA). Results are representative of 3 independent experiments (mean ± SD, P < .005).