Bim upregulated by SBHA attenuates ABT-737–induced autophagy. (A) Drug-naïve (U266) and bortezomib-resistant (PS-R) cells were treated with 500 nM ABT-737 with or without 20 μM SBHA for 16 hours, followed by immunoblotting analysis to monitor LC3 processing. (B) U266 and PS-R cells stably expressing GFP-LC3 were treated (6 hours) as described in (A), and then analyzed for GFP-LC3 puncta by confocal microscopy. Scale bar = 10 mm (magnification × 20). (C) U266 cells were treated as described in (A), after which cells were lysed in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer and subjected to immunoprecipitation (IP) with anti-Bcl-2 antibody and subsequent immunoblotting analyses using anti–Beclin-1 (Becl-1) and anti-Bim antibodies. (D) Primary CD138⁺ cells derived from a patient with relapsed MM were treated with 100 nM ABT-737 with or without 10 μM SBHA for 16 hours, after which immunofluorescence staining was performed to monitor expression of Bim or LC3 (both AlexaFluor 488, green) in CD138-phycoerythrin-positive cells. Scale bar = 10 μm; magnification ×40. (E) Tumor tissues obtained from PS-R cell mouse xenografts following the indicated treatments were subjected to immunoblotting analysis to monitor expression of Bim and LC3, as well as cleavage of caspase-9 and PARP. H, heavy chain; Ig, immunoglobulin; L, light chain.