INK128 triggers cytotoxicity in MM cell lines and primary patient cells without affecting normal cells. (A) Cytotoxicity was assessed with the MTT assay. MM cell lines (MM1S, OPM2, H929, and U266LR7) were cultured with rapamycin (5-1000nM) or INK128 (5-1000nM) for 48 and 72 hours. The average proliferation values of control, untreated samples were taken as 100%. Points indicate the means of quadruplicates of an experiment that were repeated at least twice. (B) BrdU uptake assay. MM1S and OPM2 were cultured with rapamycin (5-100nM) or INK128 (5-100nM) for 48 hours. (C) Action of INK128 in cells from patients with MM. Patient cells were plated in 6-well plates and treated with INK128 at 25, 50, and 100nM concentrations. After 24 hours, cells were stained with annexin V–FITC and 4 mAbs (CD38, CD56, CD138, and CD45), which allowed the distinction between MM plasma cells and the remaining normal BM cells, normal lymphocytes, and granulocytes. (D) INK128 (2.5-20nM) was combined with dexamethasone (250-1000nM), melphalan (2.5-7.5nM), doxorubicin (100-400nM), bortezomib (2.5-10nM), or lenalidomide (1-10 μM). After 48 hours, MTT assays were performed on MM1S and OPM2 cells. Only the most representative doses of each combination are included in the figure.