Anti-MM activity of SB. (A) MM, melanoma, leukemia, and lymphoma cell lines were treated with dimethyl sulfoxide (DMSO) control or SB at different concentrations for 48 hours, followed by assessment for cell viability using a WST assay (P < .05 for all cell lines, n = 3). IC₅₀ was calculated and presented in the table. (B) Purified CD138⁺ cells from a patient with MM were treated with DMSO or SB for 48 hours, followed by assessment for cell viability using a CTG assay (mean ± SD of triplicate cultures). (C) Normal PBMCs from healthy donors were treated with DMSO or the indicated concentrations of SB for 48 hours, and then analyzed for cell viability using a CTG assay (mean ± SD, n = 3, P value is ns). (D-E) MM.1S cells were cultured with or without pDCs or BMSCs in the presence or absence of indicated concentrations of SB for 48 hours, and then cell viability was measured by a WST assay (mean ± SD, n = 3). (F) Total BM-derived mononuclear cells from patients with MM (n = 3) were treated with Rpn10 inhibitor SB (nontoxic concentration of 0.5 μM) or DMSO control for 2 days, and multicolor flow analysis was used to assess MM cell lysis. CD138⁺ MM cells were quantified by staining with CD138-FITC Ab. Representative fluorescence-active cell sorter scatter plot showing a decrease in the number of viable fluorescein isothiocyanate–positive MM cells after treatment with SB (left) and bar graph shows quantification of CD138⁺ MM cells in the left panel (right) are shown. The fold change was obtained after normalization with control data and presented as percentage of viable cells in the presence vs absence of SB (mean ± SD, P < .05). (G) Mice bearing human MM.1S MM tumors were treated with either vehicle control or SB (20 mg/kg, intraperitoneally) 3 times weekly for 14 days. Average and SD of tumor volume (mm³) is shown vs time when tumor was measured (mean tumor volume ±SD, 10 mice per group) (left top) and Kaplan-Meier plots show survival in mice (left bottom) are shown. SB-treated mice showed increased survival vs control vehicle-treated mice. Tumor lysates from control vehicle- and SB-treated mice were subjected to immunoblot analysis using anti–caspase-3, caspase-9, K48 polyubiquitin, and β-actin (right). (H) Representative hematoxylin and eosin (HE) and immunohistochemistry stains of caspase-3, K48 polyubiquitin and Rpn10 in tumor tissue from control- and SB-treated mice. Scale bars, 100 μM.