Evidence of in situ TORC2 activation in marrow myeloma cells and possible mechanisms. (A) Percent of myeloma cells positive for phospho-AKT staining in 35 patients. (B) Example of phosphorylated AKT expression in myeloma cells within a bone marrow section. Microscopic visualization was with a Nikon eclipse E400 microscope at 400× magnification and image was captured with a Microfire camera by Optometrics using the Picture Frame program Version 6.1 (Universal Imaging). (C) MM cell lines were stimulated with IGF-1 (250 ng/mL) or IL-6 (1000 U/mL) for 30 minutes after serum depletion overnight or no serum depletion. Immunoblot assay was then performed for phospho-AKT or total AKT. (D) OPM-2 MM cells were infected with adenovirus expressing PTEN or empty vector and, 48 hours later, immunoblot assay was performed for expression of PTEN, phospho-AKT, total AKT or actin. (E) ANBL-6 MM cells stably transfected with mutant N-RAS (N), K-RAS (K) or empty vector (WT) were maintained in continuous culture in 100 U/mL of IL-6. At time 0 hour, all 3 cell lines were depleted of IL-6 for 24, 48, or 72 hours. At each time point, immunoblot assay was performed for expression of phospho-AKT or total AKT. At all time points, there was no significant difference between cell lines in percent viability or viable cell recovery.