Journal: Chinese medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1.

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Fig. 7 Taraxerone plays a protective role in sepsis-induced ALI mice through the SIRT1 pathway. The EX527 (10 mg/kg) was administered intraperitoneally 2 h before the taraxerone (0, 30 mg/kg) treatment, and the lung tissues and BALFs were collected after the CLP model was constructed 12 h later. A With H&E staining, we examined the lung tissue for any pathological changes. The bar represented 100 μm. B The breathing frequency and dynamic compliance of mice were detected by BUXCO system. C Detection of IL-1β, IL-6, TNF-α, and IL-18 mRNA levels. D ROS level in BALFs were determined using flow cytometry and analyzed with FlowJo. E Changes in the proportion of M1 macrophages in BALFs were detected using flow cytometry, and the data were analyzed using FlowJo. F Effects of taraxerone (30 mg/kg) and EX527 (10 mg/kg) on NLRP3, Cleaved Caspase-1, Pro Caspase-1, ASC, Pho-P65, Ace-P65, P65, Pho-IκB, and IκB protein levels. G–I MDA content, GSH content, and SOD activity were determined to reflect the oxidative stress levels of lung tissues. Data are presented as mean ± SD (n ≥ 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: SIRT1 polyclonal antibody (Proteintech, China) was added to the supernatant overnight at 4 °C.

Techniques: Construct, Staining, Flow Cytometry, Activity Assay

Journal: Chinese medicine

Article Title: Taraxerone inhibits M1 polarization and alleviates sepsis-induced acute lung injury by activating SIRT1.

doi: 10.1186/s13020-024-01002-z

Figure Lengend Snippet: Fig. 8 Taraxerone plays a protective role in acute lung injury by activating SIRT1. Taraxerone activates SIRT1 to block the NF-κB-NLRP3 inflammasome axis and to suppress the excessive ROS, inhibiting the M1 polarization of macrophages, and exerting a protective effect on acute lung injury mice.

Article Snippet: SIRT1 polyclonal antibody (Proteintech, China) was added to the supernatant overnight at 4 °C.

Techniques: Blocking Assay

Journal: Journal of ethnopharmacology

Article Title: Buzhong Yiqi Decoction Improves Inflammation and Oxidative Damage in Autoimmune Thyroiditis by Inhibiting Apoptosis via the SIRT1-Mediated Nrf2/NF-κB Axis.

doi: 10.1016/j.jep.2025.119967

Figure Lengend Snippet: Fig. 4. BZYQ can regulate the expression of SIRT1, NF-κB p65, Nrf2, and HO-1 in the 402

Article Snippet: The PVDF 305 membrane was blocked in 5% skim milk for 1.5 h at the room temperature and 306 incubated overnight at 4°C with specific antibodies, including SIRT1 (1:3000, 307 ProteinTech Group, Wuhan, China), NFκB-p65 Polyclonal Antibody (1:3000dilution, 308 ProteinTech Group), NFκB-p65 (phospho Ser536) Polyclonal Antibody (1:100 dilution, 309 ImmunoWay Biotechnology Company, Texas, USA), Nrf2 Polyclonal Antibody 310 (1:1000 dilution, ImmunoWay Biotechnology Company), HO-1 Mouse mAb (Mix-311 mA) (1:1500 dilution, ImmunoWay Biotechnology Company), Bax mouse 312 Monoclonal Antibody (6F11) (1:1500 dilution, ImmunoWay Biotechnology 313 Company), Bcl-2 Cytochrome c Monoclonal antibody (1:5000 dilution, ProteinTech 314 Group), Caspase-3/p17/p19 Monoclonal antibody (1:2000 dilution, ProteinTech 315 Group), and GAPDH (1:10000, ProteinTech Group).

Techniques: Expressing

Journal: Cancer Cell

Article Title: Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

doi: 10.1016/j.ccr.2008.03.004

Figure Lengend Snippet: SirT1-Related Effects of Tenovins in Mammalian Cells (A) MCF7 cells were transfected with the RGC-ΔFos- LacZ p53-dependent reporter construct as well as a control vector or an expression vector for the SirT1-363Y dominant negative mutant. All samples were also transfected with a control plasmid expressing luciferase under the control of the SV promoter. β-galactosidase activity was measured 32 hr after transfection and values were normalized using the luciferase readings. Values correspond to three independent experiments ± SD. (B) H1299 cells (p53 null) were transfected with vectors expressing p53 and mdm2 in the absence or presence of a vector expressing SirT1 (pCMV-SirT1). Cells were treated with increasing concentrations of tenovin-1 for 6 hr, and the levels of p53 and SirT1 were analyzed by western blot using DO1 and antibody 2G1-F7 (Cat. No. 05-707, Upstate), respectively. Note that pCMV-SirT1 encodes SirT1 isoform-1. Endogenous SirT1 isoform-1 was also detected in lanes 1 through 5 upon longer exposure of the blots. The band below ectopic SirT1 could correspond to a SirT1 isoform. (C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times and analyzed by western blotting using an antibody against K382-acetylated p53 (Cat. No. 614202, BioLegend) or the DO1 antibody against the N terminus of p53. PCNA was detected as a loading control. (D) H1299 cells transfected with a vector for p53 were treated for 6 hr with the indicated concentrations of tenovin-6. K382-acetylated p53 and total p53 were detected as above. (E) H1299 cells were transfected with a vector for p53 expression (upper panels) or p53R273H (lower panels) in the absence or presence of pCMV-SirT1. Cells were treated for 6 hr with the indicated concentrations of tenovin-1. K382-acetylated p53 and total p53 were detected. (F) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1, 2, and 3) or p53R273H (lanes 4, 5, and 6). Cells were left untreated (lanes 3 and 4) or treated with 10 μM (lanes 2 and 5) or 20 μM (lanes 1 and 6) tenovin-6 for 6 hr. K382-acetylated p53 and total p53 were detected, and the ratio between the amount of K382-acetylated p53 and the total amount of p53 in each lane was calculated. Note that these ratios do not correspond to the actual fraction of acetylated p53 in cells. Lanes 7, 8, and 9 correspond to loading 1/10 of the amount of protein in samples in lanes 4, 5, and 6, respectively. (G) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1 through 3) or p53R273H (lanes 4 through 6) in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5, and 6) of ectopic mdm2. In lanes 3 and 6, cells were treated for 6 hr with 10 μM tenovin-1. Total p53 was detected with DO1 antibody. β-gal expression was used as a transfection efficiency and loading control. (H) H1299 cells were treated with 10 μM tenovin-1 for the indicated times. Endogenous p14ARF was detected using a mouse monoclonal antibody (Ab-3 14P03, Neomarkers). PCNA was detected as a loading control.

Article Snippet: Human wild-type p53, p53R273H, and mdm2 expression vectors are described ( ). pCMV-SirT1 vector for SirT1 isoform 1 was obtained from Origene (Cat. No. SC127917).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Dominant Negative Mutation, Luciferase, Activity Assay, Western Blot

Journal: Cancer Cell

Article Title: Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

doi: 10.1016/j.ccr.2008.03.004

Figure Lengend Snippet: Tenovin-6 Inhibits the Protein Deacetylase Activities of Purified Sirtuins SirT1 and SirT2 (A–D) Increasing concentrations of tenovin-6 were added to purified human SirT1 (A), SirT2 (B), SirT3 (C), or HDAC8 (D) reaction mixtures. Values correspond to the average enzyme activity of three independent experiments ± SD. Estimated IC 50 values for SirT1, SirT2, and SirT3 in the assay conditions are 21, 10, and 67 μM, respectively. (E) Analysis of tenovin-6's ability to compete for binding sites with the SirT1 FdL acetylated peptide substrate. In vitro SirT1 inhibition assay was carried out with tenovin-6 at 0, 50, and 75 μM with varying FdL concentrations and a constant NAD+ concentration of 1 mM. All assays contained the same amount of DMSO (0.25%). The data is presented as a Lineweaver-Burke plot (where the x axis intercept is −1/K m and the y axis intercept is 1/V max ). Trend lines were then added to create a straight line. The trend lines for 0 μM (triangles), 50 μM (asterisks), and 75 μM (circles) tenovin-6 all have an R 2 values above 0.99. Data points are the average of triplicate experiments. (F) Analysis of tenovin-6's ability to compete for binding sites with SirT1 cosubstrate NAD+. In vitro SirT1 inhibition assay was carried out with tenovin-6 at 0, 25, and 50 μM with varying NAD + concentrations and a constant FdL acetylated peptide concentration of 200 μM. All assays contained the same amount of DMSO (0.25%). The data are presented as a Lineweaver-Burke plot. The trendlines for 0 μM (diamonds), 25 μM (squares), and 50 μM (triangles) tenovin-6 all have an R 2 value above 0.99. Data points are the average of triplicate experiments.

Article Snippet: Human wild-type p53, p53R273H, and mdm2 expression vectors are described ( ). pCMV-SirT1 vector for SirT1 isoform 1 was obtained from Origene (Cat. No. SC127917).

Techniques: Histone Deacetylase Assay, Purification, Activity Assay, Binding Assay, In Vitro, Inhibition, Concentration Assay