Journal: Nature communications

Article Title: Farnesoid X receptor activation by bile acids suppresses lipid peroxidation and ferroptosis.

doi: 10.1038/s41467-023-42702-8

Figure Lengend Snippet: Fig. 2 | Activation of FXR suppresses ferroptosis in cooperation with RXR. a Treatment of HT-1080 with 12 µM Turofexorate or Fexaramine for 7 h increases FXR expression. Levels of mRNA were normalized to GAPDH expression. Data are mean ± SD of n = 3 biological replicates. Western Blot shown is one representative experiment from n = 3. One-way ANOVA with Dunnett’s test; D = DMSO b Knockdown of FXR sensitizes cells to ferroptosis. HT-1080 were transfected with 40 nM esiRNA against FXR for 48 h and treated with 1 µM IKE for 18 h. EsiGFP- transfected cells served as a control group. Data are mean ± SD of n = 3 biological replicates; one-way ANOVA with Tukey’s test. c, d Turofexorate and Fexaramine are

Article Snippet: Compounds which were considered a hit after library screening were purchased from the following commercial suppliers: 2-TEDC (Tocris Bioscience), Anethole-trithione (ATT, Selleck Chemicals), Fexaramine (Fexa, MedChemExpress), H 89 HCl (Selleck Chemicals), Dipyridamol (Focus Biomolecules), Oxymetazoline (Santa Cruz Biotechnology), Turofexorate Isopropyl (XL335/Turo, TargetMol), ICI-89406 (Santa Cruz Biotechnology), LY231617 (Tocris Bioscience), Moxisylyte (Santa Cruz Biotechnology), L-778123 (TargetMol), AZD1208 (Tocris Bioscience), Cetaben (Santa Cruz Biotechnology), WHI-P154 (Selleck Chemicals) and Tetradecylthioacetic acid (TTA, Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, Transfection, esiRNA, Control

Journal: Nature communications

Article Title: Farnesoid X receptor activation by bile acids suppresses lipid peroxidation and ferroptosis.

doi: 10.1038/s41467-023-42702-8

Figure Lengend Snippet: Fig. 3 | FXR suppresses ferroptosis in 3D spheroid and hepatic cell models.a HT- 1080 3D spheroid models are rescued from ferroptosis by treatment with 12 µM Turofexorate or Fexaramine for 48 h. Ferroptosis was induced by 200 nM RSL3 for 48 h, 2 µM Fer-1 was used as a positive control. Representative spheroid images are shown. Mean data ± SD from n = 3 biological replicates with each 8 spheroids were quantified by high-content image analysis; one-way ANOVA with Dunnett’s test. b HepG2 cells treated with 12 µM Turofexorate or Fexaramine do not show increased levels of FXR. Levels of mRNA were normalized to GAPDH expression. Data plotted are mean ± SD of n = 4 biological replicates. Western Blot shown is one representative experiment from n = 3. One-way ANOVA with Dunnett’s test; D = DMSO. c HepG2 cells have lower ferroptosis sensitivity when treated with 250 nM RSL3, but ferroptosis can be rescued by 12 µM Turofexorate or Fexaramine. Fer- roptosis was induced for 18 h. Data plotted are mean ± SD of n = 3 biological replicates. d HepG2 cells with FXR knockout show a higher sensitivity towards

Article Snippet: Compounds which were considered a hit after library screening were purchased from the following commercial suppliers: 2-TEDC (Tocris Bioscience), Anethole-trithione (ATT, Selleck Chemicals), Fexaramine (Fexa, MedChemExpress), H 89 HCl (Selleck Chemicals), Dipyridamol (Focus Biomolecules), Oxymetazoline (Santa Cruz Biotechnology), Turofexorate Isopropyl (XL335/Turo, TargetMol), ICI-89406 (Santa Cruz Biotechnology), LY231617 (Tocris Bioscience), Moxisylyte (Santa Cruz Biotechnology), L-778123 (TargetMol), AZD1208 (Tocris Bioscience), Cetaben (Santa Cruz Biotechnology), WHI-P154 (Selleck Chemicals) and Tetradecylthioacetic acid (TTA, Sigma-Aldrich).

Techniques: Positive Control, Expressing, Western Blot, Knock-Out

Journal: Nature communications

Article Title: Farnesoid X receptor activation by bile acids suppresses lipid peroxidation and ferroptosis.

doi: 10.1038/s41467-023-42702-8

Figure Lengend Snippet: Fig. 2 | Activation of FXR suppresses ferroptosis in cooperation with RXR. a Treatment of HT-1080 with 12 µM Turofexorate or Fexaramine for 7 h increases FXR expression. Levels of mRNA were normalized to GAPDH expression. Data are mean ± SD of n = 3 biological replicates. Western Blot shown is one representative experiment from n = 3. One-way ANOVA with Dunnett’s test; D = DMSO b Knockdown of FXR sensitizes cells to ferroptosis. HT-1080 were transfected with 40 nM esiRNA against FXR for 48 h and treated with 1 µM IKE for 18 h. EsiGFP- transfected cells served as a control group. Data are mean ± SD of n = 3 biological replicates; one-way ANOVA with Tukey’s test. c, d Turofexorate and Fexaramine are

Article Snippet: After 24 h of growth, cells were transfected with a vector expressing GFP-tagged human FXR (NM_001206979, OriGene) by usingX-tremeGENEHPDNATransfectionReagent (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, Transfection, esiRNA, Control

Journal: International Journal of Endocrinology

Article Title: A Possible Mechanism of Metformin in Improving Insulin Resistance in Diabetic Rat Models

doi: 10.1155/2019/3248527

Figure Lengend Snippet: Cell viability and protein expression of FXR, MAFG, and CYP8B1 in each group by metformin treatment in HepG2 cells. (a) The effect of different doses of metformin (Met) on cell viability. Cells were treated with metformin for 96 hours, and the cell viability was measured by CCK8 assay. Results are mean ± SEM; ∗ p < 0.05 vs. control group. Protein contents were detected by western blotting and were quantified with Image J β -actin was used as a reference to verify equal loading of protein sample. The HepG2 cells were incubated with different concentrations of metformin (0, 0.5, 1, 1.5, and 2 mM) for 24 h quantification of FXR (b), MAFG (c), and CYP8B1 (d). The HepG2 cells were incubated with 1 mM metformin for various durations (0, 12, 24, or 48 h), quantification of FXR (e), MAFG (f), and CYP8B1 (g). Results are mean ± SEM; ∗ p < 0.05 vs. control group.

Article Snippet: DMEM medium (HyClone Corp.), fetal bovine serum (Sijiqing), metformin hydrochloride (Sigma, USA), CCK8 kit (Sigma, USA), FXR antibody (Biorbyt), CYP8B1 antibody (Abcam), CYP7A1 antibody (Abcam), and MAFG antibody (Abcam) were used in the experiments.

Techniques: Expressing, CCK-8 Assay, Control, Western Blot, Incubation

Journal: International Journal of Endocrinology

Article Title: A Possible Mechanism of Metformin in Improving Insulin Resistance in Diabetic Rat Models

doi: 10.1155/2019/3248527

Figure Lengend Snippet: Effects of metformin on FXR, MAFG, CYP8B1, and CYP7A1 in liver tissue induced diabetes in Wistar rats. Protein contents were detected by western blotting. FXR (a), MAFG (b), CYP8B1 (c), and CYP7A1 (e) in liver tissue were quantified using Image J. The data shown represents 3–5 independent experiments. ∗ Mean values were significantly different ( p < 0.05) compared to CON rats. # Mean values were significantly different ( p < 0.05) compared to DM rats. (d) The content of cholic acid in liver tissue was determined by ELISA. Data are expressed as mean ± SEM ( ∗∗∗ p < 0.001, ### p < 0.001).

Article Snippet: DMEM medium (HyClone Corp.), fetal bovine serum (Sijiqing), metformin hydrochloride (Sigma, USA), CCK8 kit (Sigma, USA), FXR antibody (Biorbyt), CYP8B1 antibody (Abcam), CYP7A1 antibody (Abcam), and MAFG antibody (Abcam) were used in the experiments.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Gut microbiota depletion and FXR inhibition exacerbates zonal hepatotoxicity of sunitinib

doi: 10.7150/thno.99926

Figure Lengend Snippet: FXR-autophagy pathway participated in sunitinib-induced hepatotoxicity. ( A ) H&E staining of mouse liver. ( B ) Representative image of liver. ( C-G ) Serum AST ( C ), ALT ( D ), ALP ( E ), TBA ( F ), and gall bladder weight ( G ) in FXR knockout mice. ( H-L ) Body weight ( H ), H&E staining of mouse liver ( I ), AST ( J ), ALT ( K ), and TBA ( L ) in Fxr fl/fl , Fxr ΔL , and Fxr ΔIE mice. ( M ) Correlation analysis between FXR and autophagy gene expression through RNA-Seq analysis in wide type and FXR knockout mice. ( N ) Correlation analysis between FXR and autophagy gene expression in mouse liver. ( O ) Autophagy level using cell autophagy detection assay kit after 3-MA and NH 4 Cl treatment. ( P ) Cell viability and AST level after 3-MA treatment in mouse primary hepatocytes. ( Q ) Cell viability and AST level after NH 4 Cl treatment in mouse primary hepatocytes. ( R ) FXR (FXR, BSEP, SHP, and OSTβ) and autophagy (LC3BI/II and P62) protein expression in mouse liver after sunitinib treatment. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: platelet endothelial cell adhesion molecule 1 (CD31, ab7388, Abcam, UK), CD31 (ab24590, Abcam, UK), cytokeratin 19 (CK19, ab254186, Abcam, UK), microtubule- associated protein light chain 3B (LC3B, ab192890, Abcam, UK), zonula occludens-1 (ZO-1, ab96587, Abcam, UK), FXR (NB400-153, Novus), FXR (sc-25309, Santa Cruz Biotechnology, USA), BSEP (PB0379, BOSTER), sequestonsome1 (SQSTM1/P62, ab109012, Abcam, UK), Caspase3 (ab184787, Abcam, UK).

Techniques: Staining, Knock-Out, Gene Expression, RNA Sequencing, Detection Assay, Expressing