Lenalidomide-induced ER stress triggers cytotoxicity by activating BH3 protein Bim in MM. (A) OPM2-NT and OPM2-shCRBN cells were treated with increasing concentrations of lenalidomide (10, 30, and 50 μM) for 3 days. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. The proapoptotic BH3-only protein Bim isoforms predominantly accumulated after lenalidomide treatment in CRBN-positive cells. (B) Additional CRBN-positive and CRBN-negative isogenic cell lines (OCIMY5 CRBN overexpressing, MM.1S sensitive and resistant to lenalidomide) were treated with lenalidomide for 3 days. Cell lysates were prepared and immunoblotted with the indicated antibodies. Bim activation was predominantly mediated by lenalidomide in CRBN-positive cells but not in CRBN-negative or CRBN knockdown cells. (C) Knockdown of Bim-mediated resistance to lenalidomide. Bim knockdown OPM2 clone numbers 73 and 75, and NT control cells were treated with different concentrations of lenalidomide for 4 days, and cell viability was determined with the MTT assay. Each experimental condition was performed in triplicate and repeated at least twice. (D) OPM2-NT and OPM2-shBim clone numbers 73 and 75 were treated with or without lenalidomide (10 μM) for 72 hours. Cell lysates were prepared, separated by electrophoresis, and immunoblotted as indicated. Blots are representative of 3 independent experiments.