Adherence to primary BMSCs does not protect against ONX0912-induced cytotoxicity. (A) BCWM.1 cells were cultured with control media and with ONX0912 (2.5-100nM), 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 experiments. (B-C) Primary WM bone marrow stromal cells were cultured with increasing doses of ONX0912 (10-100nM) for 8 hours. Conditioned media were collected and IL-6 (B) and IGF-1 (C) concentrations were measured by IL-6 (A) and IGF-1 (C) ELISA assays. (D) BCWM.1 cells were cultured with control media or ONX0912 (10-100nM) for 8 hours in presence or absence of primary BMSCs. Whole-cell lysates from WM cells were subjected to Western blotting using anti–p-AKT, -AKT, –p-ERK, -ERK, and –α-tubulin antibodies. (E-F) Primary WM bone marrow stromal cells were cultured with ONX0912 (20-50nM), neutralizing antibody anti–IL-6 (anti–IL-6: 0.15μg/mL), and neutralizing antibody anti–IGF-1 (anti–IGF-1: 5 μg/mL) for 8 hours. Conditioned media were collected; IL-6 (E) and IGF-1 (F) concentrations were measured by IL-6 (E) and IGF-1 (F) ELISA assays. (G) BCWM.1 cells and primary WM BMSCs were cultured either alone or in a coculture system, in presence of primary WM BMSCs, with ONX0912 (20-50nM) and anti–IL-6 (0.15 μg/mL), anti–IGF-1 (5 μg/mL), or exogenous recombinant IL6 (25 ng/mL) or IGF1 (25 ng/mL) for 48 hours. Cell proliferation was assessed using [³H]-thymidine uptake assay. All data represent mean ± SD of triplicate experiment.