Mechanisms mediating anti-MM activity of NPI-0052 plus lenalidomide. (A) MM.1S cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours. Cells were harvested, and whole cell lysates were subjected to immunoblot analysis with anticaspase-8 or anticaspase-9 Abs. FL indicates full length; and CF, cleaved fragment. Blots shown are representative of 3 independent experiments. (B) MM.1S cells were treated with indicated agents (as in panel A) in the presence or absence of biochemical inhibitors of caspase-3, caspase-8, or caspase-9, and then analyzed for apoptosis using annexin V/propidium iodide staining assay. Data are mean ± SD (n = 3; P < .005). (C) MM.1S cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours. Cells were harvested, and whole cell lysates were subjected to immunoblot analysis with anti-BIM or anti-GAPDH Abs. (D) Bar graph showing quantification by densitometry of BIM protein bands in panel C: A 2- to 3-fold increase in BIM_EL and BIM_(L+S), respectively, was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to GAPDH. (E) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 24 hours and harvested; whole cell lysates were subjected to immunoblot analysis with anti-BIM or antitubulin Abs. Blots shown are representative of 2 independent experiments. (F) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 24 hours and treated with indicated agents (as in panel A), followed by analysis for apoptosis by annexin V/propidium iodide staining. As a control, nontransfected cells were also treated with indicated drugs and similarly analyzed. Data are mean ± SD (n = 2; *P > .004). Error bars represent SD.