Fig 2: AMPK signaling blockade attenuated berberine-mediated enhancement of ATP-induced inflammasome activationA. After primed with LPS (500 ng/ml) for 4 h, J774A.1 cells were pre-treated with AMPK inhibitor compound C (C.C) (20 µ?) for 1 h before incubation with indicated concentrations of berberine for 1 h followed by co-treatment with ATP (3 m?) for 1 h. Cell death was assayed by propidium iodide staining. Data are shown as mean ± SD (n = 5). B. TG-elicited peritoneal macrophages were sequentially treated with LPS (500 ng/ml for 4h), C.C (20 µ? for 1 h), berberine (indicated doses, for 1 h) and ATP (2 mM for 30 min). Cell death was assayed by propidium iodide staining. C. TG-elicited peritoneal macrophages were treated as B. except that berberine concentration was 3 µM. Western blotting was used to evaluate the expression and secretion of IL-1ß in the cell lysates and culture supernatants (Sup), respectively. ß-Tubulin was used as a loading control for cell lysates. D. Histograms showing the relative gray values of IL-1ß in C. (n = 3). The gray value of IL-1ß band in ATP group was set as 1.0. The gray values of the other groups were calculated relative to the ATP group. *P < 0.05; **P < 0.01; ***P < 0.001; BBR, berberine.

Fig 3: RNAi knockdown of AMPKa in adult schistosomes. (A) RNAi was performed on freshly isolated adult schistosomes by electroporation in the presence of three S. mansoni AMPK a-targeting DsiRNA constructs, either separately (DsiRNA #1, DsiRNA #2, and DsiRNA #3), or in combination (all three DsiRNAs). Control parasites were electroporated with a scrambled DsiRNA control construct. AMPK a transcripts were quantified at 24, 48, and 72 h post-RNAi using quantitative real-time RT-PCR. AMPK a transcript abundance relative to the scrambled DsiRNA control were calculated. Data points represent independent biological replicates composed of 5–7 worm pairs. Bars represent mean ± standard deviation. (B) Lysates of adult schistosomes 72 h post-RNAi were prepared and equal amounts were probed by Western blot using anti-AMPK a and anti-ß-tubulin antibodies. Protein abundance was determined by normalizing the anti-AMPK a signal to the anti-ß-tubulin signal for each sample. Relative protein abundance compared to the scrambled DsiRNA samples was then calculated. WB indicates the antibodies used for Western blotting. Western blot pictured is representative of at least 3 biological replicates. (C) Representative adult schistosomes electroporated in the presence of either AMPK a-targeting DsiRNA #1, #2, #3, all 3 constructs combined, or scrambled DsiRNA were photographed 72 h post-RNAi. (D) DsiRNA-treated adult worms were phenotypically scored at 24, 48, and 72 h post-electroporation. Data shown are averages of at least 3 biological replicates. (E) The extracellular acidification rate (ECAR) of adult S. mansoni pairs 72 h post-electroporation with either all 3 AMPK a-targeting DsiRNAs or scrambled DsiRNAs were measured using Agilent Seahorse XFe96 system. Each data point is the average of at least 3 biological replicates. Data are representative of at least two independent experiments. nsP > 0.05.

Fig 4: eEF-2K-eEF2-AMPK signaling pathway is involved in the Akt activation induced by mTOR inhibitor.MDA-MB-231 or MCF-7 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF-2K, followed by treatment with 100 nM rapamycin (A) or everolimus (B) for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. ß-actin was used as a loading control. C MDA-MB-231 or MCF-7 cells were pretreated with 0.25 µM NH125 for 1 h, followed by treatment with 100 nM everolimus for 24 h. Cell lysates were analyzed by western blot for levels of indicated proteins. ß-actin was used as a loading control. D, E MDA-MB-231 or MCF-7 cells were transfected with nontargeting siRNAs or siRNAs targeting eEF2. Cell lysates were analyzed by western blot for levels of indicated proteins. ß-actin was used as a loading control. F The content of ATP in MCF-7 cells (shCtrl and sheEF2) was analyzed by the ATPlite Luminescence Assay Kit. The results are displayed as the means ± SD of triplicate measurements from one of three identical experiments; **P < 0.01, t test. G MCF-7 and MDA-MB-231 cells were transfected with nontargeting shRNA or shRNA targeting AMPK. Cell lysates were analyzed by western blot for levels of indicated proteins. ß-actin was used as a loading control. H MCF-7 and MDA-MB-231 cells (shCtrl and sheEF2) were further transfected with HA-AMPK. Cell lysates were analyzed by western blot for levels of indicated proteins. ß-actin was used as a loading control. All experiments were repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Knockdown of AMPKa1 abolishes CTRP9 protective effects in VSMCs VSMC cells were transfected with an siRNA targeting AMPa1 (siRNAa1), a control siRNA (NC), or were not transfected (-). Cells were then treated with 5 µg/ml cholesterol in the presence of CTRP9 (10 µg/ml) for 72 hrs. (A) Representative Western blot of ACTA2, phospho-AMPK (p-AMPK), total AMPK (AMPK), TLR4, MyD88, phospho-p65 (p-p65), total p65 (t-p65), LOX-1, LXR, ABCA1, ABCG1, ICAM-1 and VCAM-1 in VSMCs from each treatment. (B–L) Quantification of Western blot images. Values represent the means ± S.D. of triplicate reactions and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.