Neither growth factors nor adherence to BMSCs protect against perifosine-induced MM cell cytotoxicity. (A) MM.1S cells were cultured for 48h with control media (white bars); with 50 nM (light blue bars), 100 nM (medium blue bars), and 200 nM (dark blue bars) Dex; or with 2.5 μM (light orange bars), 5 μM (medium orange bars), and 10 μM (dark orange bars) perifosine, in the presence or absence of IL-6 (10 ng/mL). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (B) MM.1S cells were cultured with control media or perifosine (5 μM) for 6 hours. Cells were then stimulated with IL-6 (10 ng/mL) or IGF-1 (25 ng/mL) for 10 minutes. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-MEK, anti–p-ERK, anti-ERK1/2, and p-STAT3 Abs. (C) MM.1S cells were cultured with control media (white bars); and with 1.25 μM (light orange bars), 2.5 μM (medium orange bars), and 5 μM (dark orange bars) perifosine for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed by [³H]-thymidine uptake; data represent means (± SD) of quadruplicate cultures. (D) MM.1S cells were cultured with control media or perifosine (5 μM) for 6 hours in the presence or absence of BMSCs. MM.1S cells were harvested, lysed, and subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, anti-ERK1/2, and p-STAT3 Abs.