Effects of NPI-0052 plus lenalidomide on ER stress signaling, heat shock proteins, and proteasome activity. (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 ER-protein fractions were subjected to immunoblot analysis with anti-BIM or antiactin Abs. Bar graph (bottom) showing quantification of BIM_EL protein bands in immunoblot by densitometry: A 2.2-fold increase in BIM_EL isoform was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to actin. (B) MM.1S cells were treated with indicated agents (as in panel A); whole cell lysates were subjected to immunoblot analysis with anticaspase-12 or tubulin Abs. (C) MM.1S cells were treated with indicated agents (as in panel A); whole cell lysates were subjected to immunoblot analysis with anti-Hsp-70, anti-Hsp-27, anti-Hsp-90, or antitubulin Abs. Lysates from HeLA cells served as a positive control for Hsp Abs. Blots shown are representative of 2 independent experiments. (D) MM.1S cells were pretreated with or without lenalidomide for 6 hours, and then NPI-0052 was added for an additional 6 hours and harvested; cytosolic extracts were then analyzed for CT-L, C-L, and T-L proteasome activities. Results are represented as percentage inhibition in proteasome activities in drug-treated versus vehicle control. Data are mean ± SD (n = 3, P < .05).