Mechanisms of NPI-0052 plus bortezomib–induced apoptosis. (A) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–caspase-9, anti–caspase-8, anti–caspase-3, or anti–PARP Abs. FL indicates full length; CF denotes cleaved fragment. Blots shown are representative of 3 independent experiments. (B) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 24 hours in the presence or absence of pancaspase inhibitor z-VAD-fmk and then assessed for viability. Shown are the means plus or minus SD (error bar) of 3 independent experiments (P < .005). (C) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti-BIM or anti–Bcl-2 Abs. (D) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 48 hours and harvested; total protein extracts were then subjected to immunoblot analysis with anti-BIM or antitubulin Abs. Blots shown are representative of 2 independent experiments. (E) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 48 hours, followed by 12-hour of treatment with indicated agents and analysis for apoptosis by annexin V/PI staining. Error bars represent standard deviation.