Fig 1: The expression levels of p-AMPKa and FOXO3a in GC patients.a Representative images from human GC (T) and corresponding para-carcinoma (P) tissues stained with p-AMPKa and FOXO3a (data from the tissue array). Upper panel: ×40, scale bars: 500 µm. Lower panel: ×400, scale bars: 50 µm. b Expression of p-AMPKa and FOXO3a in human GC (right) and corresponding para-carcinoma (left) tissues (tissue array, n = 117). c The IHC score (staining intensity × positive percentages) for p-AMPKa and FOXO3a staining in GC and corresponding para-carcinoma tissues (tissue array, n = 117). Data are presented as mean ± SD, **** represents for p < 0.0001. d The correlation between SIRT1, p-AMPKa, and FOXO3a expression levels in GC tissues (tissue array, n = 117). e Analysis of p-AMPKa and FOXO3a expression levels in relation to the overall survival of GC patients (tissue array, n = 90). f, g Analysis of AMPKa expression levels in relation to the overall survival (f) and first progression (g) of GC patients treated with a 5-FU-based regimen from the Kaplan–Meier plotter database (209799_s_at) (n = 153). h, i Analysis of FOXO3a expression levels in relation to the overall survival (h) and first progression (i) of GC patients treated with a 5-FU-based regimen from the Kaplan–Meier plotter database (204132_s_at) (n = 153). j A schematic model showing the role of the SIRT1-AMPK/FOXO3 pathway in inhibition of chemoresistance and CSC properties of GC.
Fig 2: Positive feedback between AMPK and FOXO3.a Intracellular distribution of FOXO3a was examined by immunofluorescence staining. The AMPK activator (AICAR, 1 mM, 24 h) promoted nuclear translocation of FOXO3a, while the AMPK inhibitor (Compound C, 10 µM, 24 h) led to cytoplasmic distribution of FOXO3a. Magnification: ×400, scale bars: 50 µm. b Transcriptional activity analysis of FOXO3. Cells were pretreated with AICAR (1 mM) or Compound C (10 µM) for 2 h. Data are presented as mean ± SD (n = 3). c Real-time PCR was performed to determine the mRNA expression levels of the three subunits of AMPK. Data are presented as mean ± SD (n = 3). Fi: small interfering RNA targeting FOXO3a. Ni represents the negative control. d Western blot was performed to analyze the expression levels of AMPKα, p-AMPKα and AMPKγ. e The scheme of putative FOXO3-binding sites on the promoters of AMPKα and AMPKγ. f ChIP assays showed that FOXO3 directly interacts with the FOXO3-binding sites (mainly the second and the third putative binding sites) within the AMPKα promoter. No binding signal was detected on the AMPKγ promoter. g Luciferase activities of different AMPKα promoter constructs in GC cells treated with FOXO3a siRNAs. WT: luciferase reporter vector containing the primary AMPKα promoter, Mut-1, -2, -3: luciferase reporter vector containing the AMPKα promoter with deletion of the FOXO3-binding site 1, 2, 3, respectively. Data are presented as mean ± SD (n = 3). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.
Fig 3: Puerarin stimulated autophagy in podocytes via HMOX-1/Sirt1. (A,B) Autophagic vacuoles (autophagosomes) were detected by TEM. Representative TEM images are shown and autophagosomes are marked with red arrows. The number of autophagosomes per cell was calculated by counting the number of double-membrane organelles in 10 cells. (C) The protein expression of LC3B, p62, AMPK, and p-AMPK in podocytes was determined by western blotting. Data are expressed as the mean ± SEM (n = 3). ***P < 0.001 vs. control; ##P < 0.01; ###P < 0.001 vs. HG.
Fig 4: Autophagy is important for HG-induced podocyte injury. (A) The protein expression of p-AMPK, AMPK, LC3B, and p62 in podocytes was determined by western blotting. (B,C) Analysis of apoptosis rates by flow cytometry and quantification. (D,E) Autophagic vacuoles (autophagosomes) were detected by TEM. Representative TEM images are shown and autophagosomes are marked with black arrows. The number of autophagosomes per cell was calculated by counting the number of double-membrane organelles in 10 cells. Data are expressed as the mean ± SEM (n = 3). ***P < 0.001 vs. control; ###P < 0.001 vs. HG.
Fig 5: Immunohistochemical (IHC) staining of the proteins involved in mTOR signaling pathway. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone or CP in combination with MET or SIRO. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the IHC detection of p-mTOR (A, B) and p-AMPK (C, D) proteins. The percentage of granulosa cells with positive staining was calculated by dividing the number of positive stained cells with the total number of granulosa cells in ovarian follicles under a microscopy at ×400 magnification. One largest tertiary follicle in each section was selected to calculate the number of positively stained cells. Five sections per mice were counted from a total of 5 mice in each group (n = 5 mice). Data are expressed as the mean (%) ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. *P < 0.05, **P < 0.01. The scale bar is 50 µm. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.
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