Research Paper Volume 2, Issue 8 pp 487—503

Figure 4. (A) Anti phospho S6K immunoblot analysis of Phoenix cells treated with the mTOR/FRAP inhibitor Rapamycin (200 nM) or the AMPK agonist AICAR (1 mM) for 24 hours in serum-free, nutrient rich medium. Ctrl=untreated cells. A lower strip of the same filter was hybridized with anti-actin antiserum, to confirm equal protein loading. (B) Effect of pharmacological inhibition of the mTOR pathway on cell survival to serum deprivation under different feeding conditions. Values are mean±SD of triplicate samples. Representative of several independent experiments. (C) Immunoblot analysis demonstrating effective downregulation of mTOR/FRAP by lentiviral transduction of a targeting (sh-mTOR) or non-targeting (sh-ctrl) short hairpin RNA, and effects on the downstream signaling cascade. Cells were analyzed 24 hours after serum starvation in the indicated media (ctrl=2 g/l glucose + Aminoacids; noG= no Glucose; Rap= Rapamycin 200nM; AA- = 2 g/l glucose without glutamine and NEAA). In the anti p-S6K and anti p-4EBP1 a selective loss of the slow migrating, hyperphosphorylated band by nutrient-repleted sh-mTOR samples can be appreciated. (D) Survival assay displaying reduced mortality of sh-mTOR transduced Phoenix cells in serum-free, nutrient repleted medium. Note that nutrient-independent loss of viability was unusually high in these experimental conditions. Values are mean±SD of triplicate samples. Panel representative of two experiments performed with cells from two independent infections.