Fig 2: AMPK–mTOR may be involved in DEV-induced autophagy. (A,B) DEF cells were infected with DEV. At 48, 60, and 72 hpi, cells were harvested and the activity of AMPK and mTOR was analyzed by western blotting using the indicated antibodies. (C,D) Intensity band ratio of p-mTOR to mTOR and p-AMPK to AMPK. The difference between two group means is presented as *P < 0.05 and **P < 0.01.

Fig 3: AMPK regulates DEV induced autophagy through mTOR. (A) Effects of Compound C treatment on LC3II, phosphorylation and total levels of AMPK and mTOR. Cells were pretreated with Compound C (5 µM) or DMSO (control) for 1 h, followed by DEV adsorption for 2 h. At 48 hpi, the protein levels were measured by western blotting. (B) DEF cells transfected with GFP-LC3 for 24 h were treated with 5 µM Compound C or DEV. Formation of GFP-LC3 puncta was analyzed. (C) Titers of DEV produced by Compound-C-treated DEF cells. Cells were pretreated and infected. Virus yields are shown as TCID50/ml at 48 hpi. (D) Knockdown of AMPK affected the activity of mTOR and autophagy in DEV-infected cells. DEF cells were transfected with AMPK-specific or control siRNA, then infected with DEV. At 48 hpi, cells were harvested and western blotting was performed. (E) Virus yields in DEF cells transfected with siRNA against AMPK. Virus titres were measured using the TCID50 assay. The difference between two group means is presented as **P < 0.01.

Fig 4: Altered ATP levels seen in DKO cells but no difference in mitochondrial activity. A, respirometric analysis of oxygen consumption over time, with addition of mitochondrial modulators. B, images of live cells treated with MitoTracker (shown in green). Nuclei were stained with Hoechst (blue). Scale bars, 15 µm. C, Western blotting using antibodies against members of the five electron transport chain complexes, plus actin as loading control. D, HPLC analysis of adenine nucleotides. E, adenylate energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) derived from HPLC results in D. F, Western blotting for LKB1, total and phosphorylated AMPK (Thr172), and actin. Quantified by densitometry (right). Blot is representative of 4 experiments. ***, p < 0.001, t test. Respirometry in A, images in B and blot in C are representative of 3 experiments. Bar chart in A shows mean ± S.D. from 3 experiments. HPLC data in D and E show mean ± S.D. from 6 experiments. *, p < 0.05, ANOVA with Tukey post test. max unc., maximum uncoupled rate.

Fig 5: Effect of AWGP and KCWGP on the pathways of AMPK and PI3K/Akt/mTOR in C2C12 myotubes. C2C12 myotubes were treated with AWGP or KCWGP (0.1, 1 and 2 mg/ml) or with metformin (2.5 mM) for 45 min. Phosphorylation of (A) AMPK, (B) Akt and (C) mTOR and (B) the expression of PI3K were analyzed by western blotting. The density of each target was calculated with the expression of ß-actin. The values from the triplicate of experiments are given as the mean ± standard error of mean. *P<0.05, **P<0.01 and ***P<0.001 vs. untreated cells (N). AWGP, American wild ginseng pharmacopuncture; KCWGP, Korean cultivated wild ginseng pharmacopuncture; and M, metformin-treated cells; N, untreated cells.