Fig 2: HCV core induces the expression of MTA1, a 4E-BP1 downstream target(A) Immunoblotting analysis of MTA1 expression in lysates of HuH7 controls (WT), HuH7 stably expressing core protein variants either isolated from tumor or non-tumor cirrhotic (cT or cNT) areas and HuH9.13 harboring the HCV NS3-NS5B subgenomic replicon and its related control (HuH9.13 cured). (B) Liver proteins extracted from 9-month-old WT, transgenic mice that specifically express HCV core protein isolated from tumor and non-tumor (cT or cNT) areas (4 different mice per group) were analyzed for MTA1 expression by immunoblot.

Fig 3: Effects on key proteins at P94. (A) Images of the blots. (B–I) Protein levels were normalized to controls for p-S6 235/236 (B), p-S6 240/244 (C), p-p70 S6K (D), p-4EBP1 (E), p-mTOR (F), p-AMPK (G), p-ERK (H), and MBP (I). Each bar represents mean ± SEM for control group (n = 5), stress group (n = 4), and sleep-restricted group (n = 5). Results of ANOVA are presented in Table 3. We found no statistically significant effects of condition.

Fig 4: Combination treatment targets the mTOR pathway and alters the phosphorylation of 4E-BP1 in PDAC cellsBxPC-3, MIA PaCa-2 cells and PANC-1 cells were treated with 100 μΜ gemcitabine for 24 h and/or 100 ng/ml TRAIL for 4 h. A unit of 15 μg of total protein lysate was analysed using western blotting. a PANC-1 cell lysates were analysed with antibodies directed against total mTOR, mTOR Ser2448, Raptor, Rictor, total 4E-BP1, 4E-BP1 Ser65 and GAPDH. b BxPC-3, MIA PaCA-2 and PANC-1 lysates were analysed to look at the effect on levels and phosphorylation of 4E-BP1 at residues Ser65, Thr 37/46 and Thr70 as well as levels of eIF4E. GAPDH was used as a loading control. c The change in phosphorylation of 4E-BP1 at Ser65 in PANC-1 cells following combination treatment using TRAIL treatment for either 4 or 6 h was assessed by western blotting. PVDF membranes were immunoblotted with antibodies directed against total 4E-BP1 and 4E-BP1 residue Ser65. d The relative levels of phosphorylation of 4E-BP1 at Ser65 were quantified by scanning densitometry using ImageJ and the data are shown on the histogram as % of the values for untreated cells. All experiments were repeated three times and data are provided as means ± SEM. P-values were calculated using Student’s t-test to determine the statistical significance of the difference between untreated cells and cells treated with either gemcitabine or gemcitabine plus TRAIL (*P < 0.05 and ***P < 0.001)

Fig 5: Antagonistic regulation of mTORC1 and AMPK. Nutrient depletion (i.e., in DB) or rapamycin treatment of cells in full growth media inactivates mTORC1 but activates AMPKα. a–c Nutrient depletion induced de-phosphorylation (i.e., de-activation of mTORC1) of S6K and 4EBP1, but phosphorylation/activation of AMPK. Dictyostelium cells were grown to log-phase and transferred to DB in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Similar results were obtained in three independent experiments. d Rapamycin treatment of cells in full growth media induces de-phosphorylation of S6K and 4EBP1, but phosphorylation/activation of AMPKα. Cells were grown to log-phase and transferred to a full growth media containing 500 nM of rapamycin. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Similar results were obtained in three independent experiments. e Quantification of relative AMP/ATP ratios upon nutrient withdrawal (DB) or rapamycin treatment in full growth media (Med+Rap) in shaking culture. At times indicated, 1 × 107 cells were pelleted and lysed by freeze-thaw. The AMP and ATP levels were measured separately and values represent ratio changes as mean ± standard error. Results are from three independent experiments, with triplicates used for each independent set of experiments. Activation of AMPKα by pAICAR or 2-DG de-activated mTORC1. Log-phase cells in full growth media were treated with 1 mM pAICAR (f) or 80 mM 2-DG (g) in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Immunoblots are representative of three independent experiments. h AMPKα inhibition using dorsomorphin activated mTORC1. AX3 cells were transferred to DB in the presence or absence of 40 μM dorsomorphin in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Immunoblots are representative of three independent experiments