ABC294640 inhibited cell proliferation and induced apoptosis in MM cells. (A) Dose-dependent inhibition of cell proliferation by ABC294640. Six different MM cell lines were treated with various concentrations of ABC294640 for 48 hours and cell proliferation was measured by MTT assay (mean ± standard error of the mean [SEM] of 1 of 3 separate sets of experiments). (B) Time course of proliferation inhibition by ABC294640. Six different MM cells lines were treated with 30 µM of ABC294640 or dimethylsulfoxide (DMSO) for various durations. Cell proliferation was analyzed by MTT assay at the time-points indicated (mean ± SEM of 1 of 3 separate sets of experiments). (C) Cytotoxic effects of ABC294640 on MM cells. OPM1 cells were treated with 30 µM of ABC294640 or DMSO and total live cells were quantified at the time-points indicated (mean ± SEM of 3 separate sets of experiments). (D) Increased Annexin V⁺ cells by ABC294640. OPM1 cells were treated with 30 µM of ABC294640 or DMSO for 16 hours and cells were stained with Annexin V and 7-amino-actinomycin D (AAD) and analyzed by flow cytometry. Data shown were representative of 3 separate sets of experiments. (E) Caspase 3 activation. OPM1 cells were treated with 30 µM of ABC294640 or DMSO for 16 hours and cells were then fixed, permeabilized, and stained with caspase3 antibody. (F) Caspase 9 activation and PAPR cleavage. OPM1 cells were treated with 30 µM of ABC294640 (indicated as [A]) or DMSO control (indicated as [D]) for 3, 6, 9, and 12 hours, and analyzed for PARP and cleaved PARP, full length caspase-9, and cleaved caspase-9 by western blot analysis. β-actin was used as a loading control (data were representative of 3 separate sets of experiments). (G) Inhibition of primary human CD138⁺ myeloma cells by ABC294640. Primary human CD138⁺ cells were freshly isolated using CD138 enrichment kit from the BM aspirates of myeloma patients and cultured in triplicate at 1 × 10⁴ cells in 100 μL of RPMI1640 medium supplemented with 2 mM Glutamax and 10% fetal calf serum containing DMSO control or various concentrations of ABC294640 for 24 hours at 37°C under 5% CO₂. Cell proliferation was then measured by MTT assay. OPM1 cells were similarly treated for comparison (n = 3; Sample ID #7244, #7452, and #7476).