Fig 1: PTE regulated bile metabolic related gene expression via SIRT1/FXR signaling pathway (A) A representation of the computationally predicted cluster of PTE bound to SIRT1 (B–C) Primary hepatocytes isolated from WT mice treated with PTE or SRT1720 (SIRT1 agonists). B: The activity of SIRT1 determined by SIRT1 Activity Assay Kit, n = 5. C: Whole cell lysates from primary hepatocytes were immunoprecipated with FXR antibody and immunoblotted using Ac-Lys antibody, n = 5 (D) Western blot analysis was used to confirm the expression of FXR and β-actin in primary hepatocyte cells, and qPCR analysis of Bsep were shown in the right of panel D, n = 5 (E–G) Primary hepatocytes cells treated with LPS, si-SIRT1, and PTE shown in the panel E. E: Whole cell lysates from primary hepatocytes cells were immunoprecipated with FXR antibody and immunoblotted using Ac-Lys antibody. F: western blot analysis was used to determine protein level of FXR, SIRT1, and β-actin in primary hepatocytes cells, the quantitative results were shown in the right panel, n = 5. G: qPCR analysis of Cyp7a1, Cyp8b1, Ntcp, Oatp, Abcc4, Bsep, and Abcc2 in primary hepatocytes isolated from WT mice, n = 5. *p < 0.05. The data represent the mean ± SD. Abbreviations: IB, immunoblotting; IP, immunoprecipitation.
Fig 2: SIRT1 expression in LPS-treated k562 cells. a qPCR was used to measure the mRNA expression of SIRT1 at 0, 4, 8, and 12 h after LPS treatment in k562 cells. b, c WB was performed to detect SIRT1 protein levels in k562 and KU812 cells after LPS treatment. d SIRT1 subcellular localization in LPS-treated k562 cells detected using IFA. SIRT1 was stained to red using anti-SIRT1 antibody (red), whereas nuclear DNA was counterstained blue using DAPI (blue). The merged image displays the nuclear localization of SIRT1. e Cell fractionation assay showed the location of SIRT1 in nuclear and cytoplasmic fractions. f SIRT1 activity was detected in the whole cell lysate. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 relative to the control
Fig 3: ROS generation is responsible for SIRT1-mediated inflammation in k562 cells. Here, k562 cells were subjected to 5 μM of NAC and 10 μg/mL of LPS for 4 h. a ROS production in k562 cells was reflected by DCF fluorescence intensity. b–d qPCR was performed to determine the mRNA levels of SIRT1, TLR4, and p65 in k562 cells. e WB was performed to determine the protein levels of SIRT1, TLR4, and p65 in k562 cells. f qPCR was performed to determine the mRNA levels of IL-1β, IL-6, and TNF-α in k562 cells. Gene expression in each group was first normalized to the GAPDH gene, and then normalized to the data of the control group. g SIRT1 subcellular localization in LPS-treated k562 cells detected viaIFA. SIRT1 was stained to red through anti-SIRT1 antibody (red), whereas nuclear DNA was counterstained blue through DAPI (blue). The merged image displays the nuclear localization of SIRT1. h Cell fractionation assay showed the location of SIRT1 in nuclear and cytoplasmic fractions. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 and #P < 0.05, ##P < 0.01 relative to the control and LPS groups, respectively
Fig 4: Hcy increases apoptosis in human chondrocytes. Cells were treated with Hcy (100 μM) for 24 h. For overexpression, cDNAs of SIRT1, AMPK and PGC-1 were transfected for 48 h before Hcy stimulation. (A-C) Representative Western blots and quantification data showing that Hcy treatment upregulated pro-apoptotic protein (Bax) and downregulated anti-apoptotic (Bcl-2) protein as a result of inhibition of SIRT1/AMPK/PGC-1α. (D) In DPI intervention group, DPI was pretreated for 1 h before Hcy stimulation. A TUNEL assay was used for investigation of apoptosis. (Data are presented as the mean±SD of three different experiments. *p < 0.05 compared with untreated control cells. & p < 0.005 compared to Hcy-treated group).
Fig 5: SIRT1 activation upregulates the expression of ZO-1 and collagen I. (a) Representative immunofluorescent staining images of ZO-1 (red) and nuclei (blue) of mouse hearts. Quantified ZO-1 expression was normalized to the WT group (Scale bars, 200 µm). (b) Representative Masson’s trichrome staining of high-magnification sections of the hearts of mice. (c) Cardiac collagen I protein expression were determined by Western blotting. α-tubulin was used as the internal control. Densitometric analysis of collagen I protein level is shown. The data are presented as means ± SEM of at least three independent experiments. (n = 5 mice in each group; * p < 0.05 vs. WT mice. # p < 0.05 vs. CRIF1 EKO mice).
Supplier Page from Abcam for SIRT1 Activity Assay Kit (Fluorometric)