Fig 1: Metformin's inhibition on AGEs-induced NF?B signaling is AMPK dependent. BMDMs were divided into 4 groups: control, AGEs, AGEs + MET, and AGEs + MET + CC group. In AGEs group, cells were cultured with AGEs at 200 mg/L for 60 min; in AGEs + MET group, cells were pretreated with metformin for 60 min and then cultured with AGEs at 200 mg/L for 60 min; in AGEs + MET + C-C group, cells were pretreated with Compound C, an AMPK inhibitor, at 5 µM for 60 min, and then they were treated with metformin at 2.0 µM for 60 min followed by AGEs at 200 mg/L for 60 min; in control group, cells were cultured with BSA at 200 mg/L for 60 min. p65 nuclear translocation of each group was evaluated by immunofluorescent staining. Primary antibodies against p65 and Cy3 (red) labeled secondary antibodies were used to detect p65; DAPI (blue) was used to stain the nucleus. Bar graphs represent the results (mean ± SD) of three independent experiments. Bar = 50 µm. One-way ANOVA was applied and the overall ANOVA was significant. ** p < 0.01 and *** p < 0.001 when compared between selected groups.
Fig 2: ATL-III inhibits the invasion, migration and EMT process of IEC-6 cells induced by TGF-ß1 by activating the AMPK signaling pathway. (A) Cell migration, as determined using a wound healing assay. (B) Cells invasion was detected using a Transwell assay. Magnification, ×100. Histograms of cell (C) migration and (D) invasion rates. (E) Expression levels of MMP9 and proteins associated with EMT, as detected via western blotting. (F) N-cadherin and (G) E-cadherin expression was detected using an immunofluorescence assay. Magnification, ×200. ***P<0.001 vs. control group; ###P<0.001 vs. TGF-ß1 group; ?P<0.05, ??P<0.01 vs. TGF-ß1 + ATL-III (20 µmol/l) group; n=3. ATL-III, atractylenolide III; EMT, epithelial-mesenchymal transition; AMPK, AMP-activated protein kinase; ZO-1, zonula occludens-1.
Fig 3: Effects of EMPA on AMPK/SIRT1 and oxidative stress in cultured human podocytes. (A) Protein levels of SIRT1, AMPK, p-AMPK, Foxo1 and SOD2 in podocytes determined by Western blot analysis. (B) Densitometric evaluation of Western blots for SIRT1 (n = 3), (C) p-AMPK (n = 3), (D) Foxo1 (n = 3) and (E) SOD2 (n = 3). (F) Representative immunofluorescence images of SIRT1, p-AMPK, Foxo1 and SOD2 in podocytes. Scale bar: 100 µm. Data are expressed as the mean ± SEM and were analyzed by ANOVA with LSD post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: Metformin inhibits NIH 3T3 cell migration by activating AMPK/mTOR in a concentration-dependent manner. (A,B) NIH 3T3 cells in the upper chamber were incubated in the presence of different concentrations of metformin (2.5 mM, 5 mM, 10 mM) for 24 h and the number of migrating cells was observed in five random fields under a 100× microscope. Scale bar = 100 µm. Cell migration was quantified using ImageJ/Fiji software. Values represent the means ± SEs of the numbers of migrating cells from three independent experiments. (C-G) Relative band densities of pmTOR/mTOR, pAKT/AKT, MMP2, and MMP9 are quantified in the bar graphs. All the experiments were performed in triplicate and repeated on at least three occasions. Data are shown as means ± SDs. * p < 0.05, ** p < 0.01, and *** p < 0.001. Control: NIH 3T3 cells, LPS: NIH 3T3 cells treated with 1 µg/mL LPS for 24 h, Met (2.5 mM): NIH 3T3 cells treated with 2.5 mM metformin for 24 h, Met (5 mM): NIH 3T3 cells treated with 5 mM metformin for 24 h, Met (10 mM): NIH 3T3 cells treated with 10 mM metformin for 24 h.
Fig 5: miR-29c-3p could partly rescue the changes of glycolytic capacity and the expression of the phenotype-associated proteins caused by MTFR1 in LUAD cells. (A–C) miR-29c-3p could rescue the effect of MTFR1 on the glycolytic capacity of LUAD cells. (D–F) miR-29c-3p could reverse MTFR1-induced alterations in the expression of proteins associated with cell proliferation, apoptosis, EMT and the AMPK/mTOR signalling pathway in LUAD cells. Data were articulated as the mean ± standard deviation (SD; *p < 0.05, **p < 0.01).
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