Fig 2: GSK3ß activity potentiates the phosphorylation of mTORC2 effectors in vivo under basal conditions. a The hippocampi from wildtype (n = 6) and GSK3ß S9A (n = 5) mice were isolated and lysed. Western blot was used to determine the levels of P-S235/S236-rpS6, P-S240/S244-rpS6 and rpS6. Tubulin is shown as a loading control. b WB analysis results. c Western blot was used to determine the levels of P-S473-Akt, Akt, P-Thr346-NDRG1 and NDRG1. Tubulin is shown as a loading control. d WB analysis results. Error bars indicate SEM. **p < 0.01, *p < 0.05 (unpaired t test)

Fig 3: Feeding the mice with the docosahexaenoic acid-enriched diet prior to 48 h fasting prevented decreased Akt phosphorylation and increased adenosine monophosphate-activated protein kinase-phosphorylation in the fasted tibialis anterior muscle. Representative Western blots using antibodies against total protein or specific phosphoprotein (i.e. PSer473-Akt, PThr172-adenosine monophosphate-activated protein kinase, PThr32-FoxO3, and PSer240/244-rpS6) are shown in (A). After quantification, the P-Ser473Akt/Akt (B), P-Thr172AMPK/AMPK (C), P-Thr32FoxO3A/Foxo3A (D), and P-Ser240/244rpS6/rpS6 (E) ratios were calculated in mice fed either the control or the docosahexaenoic acid-enriched diet. Data are means ± SEM (n = 6/group). Statistical differences were assessed by analysis of variance. Bars with different superscript letters are statistically different.

Fig 4: Cell Viability and Expression of Markers during Treatment of Cells with Biguanides(A) Cell viability by flow cytometry (using DAPI to label dead cells) for three established T-ALL cell lines (M100, F04, and F15) and for primary lymphoma cells derived from mice with T-cell-specific deletion of PTEN and/or AMPK after incubation for 24 h in the indicated concentrations of metformin. Results (means ± SEM, n = 8) were fitted as described in the STAR Methods.(B) As shown in (A) but with phenformin.(C) Phosphorylation of AMPK, ACC and RPS6 after 1 hr treatment with different concentrations of phenformin (n = 2).(D) Western blotting of various markers in tPTEN-/- T-ALL cell lines treated for 24 h with 1 mM metformin or 10 µM phenformin (n = 2).(E) Effect of incubation with phenformin (1 h) on mTORC1 signaling (pRPS6:RPS6 ratio) in T-ALL cells with or without AMPK. Results are means ± SEM (n = 4); significance of differences from vehicle control are indicated by asterisks (ns, not significant).

Fig 5: DGK? participates in the rapamycin-induced feedback loop in SW480 cells. (a) Cells were treated with DMSO, R59949, rapamycin or the two inhibitors for 24 h. Total cell DAG was determined in an in vitro radioenzyme assay. A representative autoradiogram (top) and mean±s.e.m. of at least three independent experiments are shown (bottom). Data analyzed using analysis of variance (ANOVA). ns, non significant; **, significant (P<0.01); ***, significant (P<0.001). (b) Cells were treated as in (a), and DGK activity determined in membrane fractions. Relative DGK activity is indicated beneath a representative autoradiogram of three independent experiments. Activity of vehicle-treated cells=100. (c) Cells in exponential growth were rapamycin treated (100 nM) for indicated times, and S6, PKCa, AKT, PKD phosphorylation and PKC activity were tested by western blot in total cell lysates. The expression of the corresponding total protein was determined in separate blots to avoid interference between the distinct antibodies and tubulin was used as loading control in the same gel to discard changes in protein expression because of rapamycin treatments. Phosphorylation for each protein was normalized to total expression of the corresponding protein, and to control cell values at time 0 (1.0). (d) Control and DGK?-silenced cells were treated with rapamycin (100 nM) or torin (250 nM) for 24 h and DGK activity determined. Relative DGK activity is indicated beneath a representative autoradiogram of three independent experiments. Activity of vehicle-treated cells=100. (e) PKD and rpS6 phosphorylation was determined in total lysates of cells stably transfected with siRNA control or specific for DGK? and rapamycin treated (100 nM; 24 h). Phosphorylation was determined as in (c), normalized to PKD and S6. Tubulin is shown as loading control. In all cases, blots shown for one representative experiment (n?3).