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.
