Bim acts as a determinant of ABT-737 sensitivity in Bim^hi MM cells. (A) U266 (upper left), RPMI8226 (upper right), and MM.1S (lower) were treated with the indicated concentrations (nM) of ABT-737 for 24 or 48 hours, after which expression of Bim and Mcl-1 was monitored by immunoblotting analysis. (B) U266 cells stably transfected with Bim shRNA (left upper panels) were treated with 750 nM ABT-737 for 72 hours, followed by immunoblotting analysis for monitoring caspase-9 (Casp-9) cleavage (left lower panels), Bax translocation (to mitochondria, right upper panels), and cytochrome c (Cyt c) release (to cytosol, right lower panels). Bak was probed for loading control of the mitochondria-enriched fraction. The vertical lines indicate the splice site in the composite image derived from a single blot. (C) RPMI8226 cells were stably transfected with HA-tagged human full-length Bim (inset, using an anti-HA antibody [αHA]) or an empty vector (EV). Cells were then exposed to 500 to 750 nM ABT-737 for 48 hours and 72 hours, after which the percentage of apoptotic cells was determined by flow cytometry. Numbers indicate P values. (D) Bortezomib-resistant U266 cells (PS-R) were transiently transfected with an empty vector or HA-tagged Bim construct. After 48 hours, untreated cells were lysed and subjected to immunoblotting analysis for HA-Bim (inset, using αHA) or flow cytometry to determine the percentage of apoptotic (annexin V⁺) cells. IB, immunoblot; FITC, fluorescein isothiocyanate; L.E., long exposure; M.E., medium exposure; S.E., short exposure; UT, untreated.