Fig 2: Down-regulation of Angptl4 expression by AMPK is likely mediated via inhibition of PPARγ-mediated transcription of Angptl4.(A) Angptl4 mRNA in differentiated T37i adipocytes pre-incubated with 0.5 μg/mL actinomycin D (ActD) or DMSO control for 1 hr and treated with 1 mM AICAR or control for 3 hr. (B) Angptl4 mRNA in differentiated T37i or BA adipocytes treated with DMSO control, 5 μM rosiglitazone, 10 μM Wy14643, or 5 μM GW742 (BA adipocytes) or 5 μM GW501516 (T37i adipocytes) for 6 hr. (C) Immunoblot for ANGPTL4 in differentiated T37i adipocytes treated with 5 μM rosiglitazone (Rosi), 10 μM Wy14643 (Wy), and 5 μM GW501516 for 24 hr. (D) Angptl4 mRNA in differentiated T37i cells treated with DMSO control, 5 μM rosiglitazone (Rosi), 1 mM AICAR or both rosiglitazone and AICAR for 6 hr. (E) Relative luciferase activity of HepG2 cells transfected with pGL3-Angptl4, pSG5-Pparγ, pSG5-Rxr, pcDNA-P300HA vectors, as indicated and treated 16 hr post-transfection with 5 μM rosiglitazone, 1 mM AICAR or both compounds for 9 hr. Data are represented as ± SD. (F) Immunoblot of P300 and phospho-P300 (Ser-89) in differentiated T37i brown adipocytes treated with control or 1 mM AICAR for 3 hr. *Statistically significant compared to control samples or between indicated samples according to Student’s t-test (p<0.05). Error bars represent ± SEM, unless otherwise specified.DOI: http://dx.doi.org/10.7554/eLife.08428.014

Fig 3: In vitro activity of human recombinant AMPK. (A) Fluorometric enzymatic assay with the human recombinant AMPK isoform α1β1γ1. This assay evaluated the ability of compound 8c to activate AMPK isoform α1β1γ1and, in turn, phosphorylate the synthetic substrate peptide AMARAASAAALARRR. The compound was evaluated at 30 μM using AMP as a positive control and the Abbott activator A-769662 as positive reference compound. (B) Activity of compound 8c on isolated AMPKα1 1–550 fragment. (C) Co-incubation assay with the human recombinant AMPK isoform α1β1γ1. The compound 8c was evaluated at 30 µM alone or in the presence of different concentrations of A-799662 from 0.3 µM to 30 µM. AMP was used as a positive control for the assay and A-799662 (0.3-3-30 µM) was used as a positive reference compound. Data are the mean ± S.D. of four experiments. **p < 0.01 and ***p < 0.001 compared by the Student’s t-test.

Fig 4: Effect of 2-oxindole fluorinated derivatives on AMPK activity in prostate cancer cells. (A) PC-3, DU145 and LNCaP cells were treated with 25 µM of the selected 2-oxindole fluorinated derivatives for 1 hour. Levels of the phosphorylated AMPK (pAMPK), phosphorylated ACC (pACC) and their total forms were determined by Western blot. Upper panel, a representative Western blot of three different experiments. β-tubulin (β-Tub) serves as a loading control. (B) PC-3, DU145 and LNCaP cells were treated with 25 µM of compound 8c, 25 µM of AMP or 25 µM of A-769662 for 1 hour and processed as above. Lower panels, densitometric analyses of bands represented as the mean of the ratio pAMPK/AMPK or pACC/ACC ± SD of three different experiments. *p < 0.05 compared with the controls by one-way ANOVA followed by Fisher’s LSD test; #p < 0.05 compared with 2-oxindole fluorinated derivates or with the AMPK activators AMP or A769662 by one-way ANOVA followed by Tukey test.

Fig 5: Cordycepin increases AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation (A) and AMPK activity (B). Cells were treated with AICAR (1 mM), cordycepin (0.1–10 μM) or cordycepin + compound C (10 μM + 40 μM) for 1 hr. Phosphorylation of AMPK (pThr172-AMPK) and its substrate ACC (pSer79-ACC) were detected by western blot analysis. The mean grey values of each lane in the western blot photos were quantified by ImageJ 4.1 software and the ratios of p-AMPK/t-AMPK and p-ACC/t-ACC were calculated. The AMPK activity was determined by the SAMS peptide assay. The data depict the means ± SEM of at least three experiments. Asterisks represent statistically significant differences from the oleic acid–treated control group (*P < 0.05, **P < 0.01, ***P < 0.001). cpn: cordycepin; C.C; compound C.