Neither growth factors nor adherence to BMSCs protect against NPI-0052–induced cytotoxicity. (A) BCWM.1 cells were cultured with control media, and with NPI-0052 (N) (2.5-20 nM), with and without bortezomib (B) (10 nM) for 48 hours, in the presence or absence of BMSCs. Cell proliferation was assessed using [³H]-thymidine uptake assay. All data represent mean (± SD) of triplicate experiment. (B) BCWM.1 cells were cultured with control media or NPI-0052 (2.5-20 nM), with and without bortezomib (10 nM) for 48 hours, in the presence or absence of IL-6 (25 ng/mL) (10 μM). Proliferation was assessed by thymidine uptake assay. (C) BCWM.1 cells were cultured with control media or NPI-0052 (10 nM) with and without bortezomib (10 nM), for 8 hours. Cells were then stimulated with IL-6 (25 ng/mL) for 10 minutes. Whole-cell lysates were subjected to Western blotting using anti–p-AKT, anti-AKT, anti–p-STAT3, and anti–α-tubulin. (D) Colony-forming cell assay. Negative fraction after CD19⁺ selection of bone marrow mononuclear cells was cultured using methylcellulose semisolid technique in absence or presence of NPI-0052 (10 nM, 20 nM) either alone or in combination with bortezomib 10 nM. Erythroid burst-forming units (BFU-Es), granulocyte-macrophage colony-forming units (CFU-GMs), macrophage colony-forming units (CFU-Ms), and granulocyte, erythrocyte, monocyte, macrophage colony-forming units (CFU-GEMMs) were counted at day 14. All experiments have been done in triplicate.