Mechanisms of b-AP15–induced MM cell death. (A) MM.1S cells were treated with DMSO or b-AP15 (100 nM) for 24 hours, followed by analysis for apoptosis with Annexin V/PI double staining (mean ± SD; n = 3; P < .001). (B) MM.1S cells were treated with DMSO or b-AP15 for 12 hours; protein lysates were subjected to immunoblotting with anti-PARP, caspase 3, caspase 8, or caspase 9 antibodies. (C) MM.1S cells were treated with b-AP15 (100 nM) for 12 hours, followed by measurement of caspase 8 and caspase 9 enzymatic activity (mean ± SD; n = 3; P < .001). (D) MM.1S cells were incubated with or without pan-caspase inhibitor for 1 hour and then treated with DMSO or b-AP15 (150 nM) for an additional 24 hours, followed by assessment of cell viability using MTT assay (mean ± SD; n = 3; P < .005 for b-AP15 alone vs b-AP15 plus pan-caspase inhibitor). (E) MM.1S and KMS-11 cells were treated with DMSO or b-AP15 (100 nM) for 48 hours and fixed in 70% ethanol. After washing with phosphate-buffered saline, cells were stained with PI, and DNA content of cells was analyzed using fluorescence-activated cell sorter. (F) MM.1S and KMS-11 cells were treated with DMSO or b-AP15 (100 nM) for 24 hours; protein lysates were subjected to immunoblotting with anti-CDC25C, CDC2, cyclin B1, or β-actin antibodies. (G) MM.1S MM cells were treated with DMSO or b-AP15 (100 nM) for the indicated times; protein lysates were subjected to immunoblotting with anti–p-eIF2α, p-IREα, CHOP, or β-actin antibodies. (H) MM.1S MM cells were treated with DMSO or b-AP15 (100 nM) for 24 hours; cytosolic and nuclear proteins were prepared with NE-PERNuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL) and subjected to immunoblotting with anti–XBP-1 and Lamin A/C antibodies. FL, full length of the protein; CF, cleaved form of the protein; s, splicing form; u, unsplicing form.