Mechanisms of SB-induced MM cell death. (A) AMO1 and MM.1S cells were treated with DMSO control or SB at the IC50 concentration for 24 hours; protein lysates were subjected to immunoblot analysis using anti-K48 polyubiquitin or anti–β-actin Abs. (B) MM.1S cells were treated with DMSO, SB, or bortezomib (BTZ) for 3 hours; protein lysates were analyzed for proteasome activities. The percentage of proteasome activity was normalized to a DMSO control (mean ± SD, n = 3). (C) Recombinant human proteins USP1, USP2, USP7, USP21, USP28, UchL1, UchL3, and UchL5 were incubated with SB for 30 minutes at 37°C and then analyzed for DUB activity (mean ± SD, n = 3). (D-H) Indicated cells were treated with DMSO or SB; protein lysates were then subjected to immunoblotting using ER stress-related antibodies against p-eIF2α, PERK, BiP, and calnexin (D); caspase-related antibodies against caspase-3, caspase-8, caspase-9, and PARP (G) (caspase-3/-9 (AMO1, MM.1S, ANBL6-BR) have same actin reprobe; PARP/caspase-9 (AMO1-CFZ.R) have same actin reprobe); and autophagy-related antibodies against LAMP2, p62, LC3, or β-actin (H); treated cells were subjected to cell cycle analysis (E) or apoptosis analysis (F). CL, caspase-like proteasome activity; CTL, chymotrypsin-like proteasome activity; TL, trypsin-like proteasome activity.
