JNK is involved during PK + bortezomib-induced apoptosis in MM cells. (A) SP6000125 (SP), inhibitor of JNK, abrogates PK + bortezomib–induced release of Smac or cyto-c. MM.1S cells were treated with PK (50 μM) + bortezomib (2 nM) in the presence or absence of SP600125 (SP) and harvested at 24 hours. Cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti–Cyto-c or anti-Smac (top and middle panels) Abs. As a control for equal loading of proteins, filters were also reprobed with antitubulin Ab (bottom panel). Blots are representative of 3 independent experiments with similar results. (B) Overexpression of DN-JNK enhances resistance to PK + bortezomib. Cells were transiently transfected with cDNA expression construct containing GFP with either DN-JNK (•) or empty vector (▪). Following transfections, GFP-positive cells were selected by flow cytometry; treated with PK (50 μM) + bortezomib (2 nM) for 24 hours, 48 hours, or 72 hours; and analyzed for cell viability by MTT assay (P = .05, as determined by one-sided Wilcoxon rank-sum test). Error bars indicate standard error.