ABC294640 acted synergistically with Bcl-2 inhibitor in inhibiting myeloma cell growth and induced myeloma cell apoptosis in the presence of BM stromal cells. (A) ABC294640 did not affect Bcl-2 expression. MM cells (OPM1, RPMI-8226, MM.1S, and U266) were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours, and whole cell lysates were prepared and analyzed for Bcl-2 expression by western blot analysis. (B) Combination of ABC294640 and ABT-737 led to enhanced inhibition of cell proliferation. OPM1 cells were treated with various concentrations of ABT-737 in the absence or presence of ABC294640 (15 µM) for 48 hours and cell proliferation was measured by MTT assay. (C) FA-CI plots showing the synergistic effect of ABC294640 and ABT-737. Fa-CI plots for OPM1 cells revealed a synergistic inhibitory effect for ABC294640 15 μM and ABT-737 at 0.1 µM (indicated as 1), 0.3 µM (indicated as 2), and 1 µM (indicated as 3). In the Fa-CI plot, the line (combination index = 1) indicate an additive reaction between the 2 substances. Values below this line imply synergism. (D) ABC294640 induced myeloma apoptosis in the presence of BM stromal cells. GFP-expressing OPM1 cells were cultured on the monolayer of HS5 BM stromal cells and were treated for 8 hours with 30 µM of ABC294640 or DMSO. The cells were stained with Annexin V and 7-amino-actinomycin D (AAD) and Annexin V⁺ apoptotic cells were gated on GFP-positive OPM1 cells.