siRNA-mediated inhibition of Rictor and Raptor in MM cell lines. MM1S and OPM2 cells were transfected with scramble probe, Raptor, Rictor, or both siRNAs. (A) Whole-cell lysates were subjected to Western blot analysis using anti-Raptor, anti-Rictor, anti–p-Akt, anti–p-Erk, anti-Erk, and anti–α-tubulin Abs. (B) Cells were treated with rapamycin 25nM for 48 hours and apoptosis was assessed with annexin V. (C) MM1S and OPM2 knockdown cells were cultured with rapamycin 25nM for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed using the BrdU uptake assay. (D) Adhesion assay with MM1S and OPM2 knockdown cells to BMSCs in the presence or absence of rapamycin 25nM. All data represent means ± SD of quadruplicate experiments. (E) MM1S and OPM2 cells were cultured with control medium, rapamycin (5-100nM), or INK128 (5-100nM) for 5 hours. Whole lysates were subjected to Western blot analysis using p-rS6 Ser235/236, p-4EBP1 Thr 37/46, and α-tubulin.