GW4064 was purchased from Cayman Chemical Company (Catalog Number 10006611) and stored at -20°C in 20mg aliquots. Preparation of GW4064 solution for animal administration was performed on the day of injection. 20mg of GW4064 was first dissolved in 1000μL of 99.5% DMSO, then diluted with 1985μL of water to reduce DMSO concentration. 16μL of TWEEN 80 was then added to return GW4064 to solution. Vehicle solution was prepared with DMSO, water, and TWEEN 80 without GW4064. 50mg/kg of GW4064 solution, and equivalent volumes of vehicle, were administered to mice via intraperitoneal injection twice a week.
Gw4064
Manufactured by Cayman Chemical
Sourced in United States
GW4064 is a synthetic compound that acts as a selective agonist for the farnesoid X receptor (FXR). FXR is a nuclear receptor that plays a key role in regulating bile acid, lipid, and glucose metabolism. GW4064 can be used in research applications to study the physiological functions of FXR and its potential therapeutic implications.
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Lab products found in correlation
Sml2148, by Merck Group (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Scramble sirna, by Horizon Discovery (1 mentions)
Bile acid, by Merck Group (1 mentions)
Gfp antibody, by Merck Group (1 mentions)
Cholestyramine, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ire1α antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Huh 7 cell line, by Health Science Research Resources Bank (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
Gw7647, by Cayman Chemical (1 mentions)
Gw1929, by Cayman Chemical (1 mentions)
Bmp 2, by Miltenyi Biotec (1 mentions)
Naphthol as mx phosphate, by Nacalai Tesque (1 mentions)
9 cis retinoic acid, by Fujifilm (1 mentions)
Lithocholic acid, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
8 protocols using gw4064
1
GW4064 Preparation and Administration
2
Bile acid signaling pathway regulation
3
Investigating HCC Cell Signaling Pathways
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Human HCC HepG2 cell line was obtained from ATCC (Manassas, VA), and Huh7 cell line was obtained from Health Science Research Resources Bank (JCRB0403, Osaka, Japan). Cells were cultured in DMEM media with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a 5% CO2 setting. All kinase-selective small chemical inhibitors for MAPK, MEK1/2, PI3K JNK, and p38MAPK, as well as GW4064, were purchased from Cayman Chemicals (Ann Arbor, MI). Chemicals were initially dissolved in DMSO and then diluted with cell culture media into the final concentration at a 1000-fold dilution. Chemical treatment time and final concentrations was indicated in the figure legends.
Antibodies for NR0B2 (clone N2C3) were obtained from GeneTex (Irvine, CA). Antibodies for ERK (clone 137F5), phosphor-ERK (clone D13.14.4E), AKT (clone 40D4), phospho-AKT (clone D9E), FXR (clone E4B8P), and Actin (clone E4D9Z) were purchased from Cell Signaling Tech (Danvers, MA). HRP-conjugated secondary antibodies and luminol reagents were purchased from Santa Cruz Biotech (Dallas, TX).
Antibodies for NR0B2 (clone N2C3) were obtained from GeneTex (Irvine, CA). Antibodies for ERK (clone 137F5), phosphor-ERK (clone D13.14.4E), AKT (clone 40D4), phospho-AKT (clone D9E), FXR (clone E4B8P), and Actin (clone E4D9Z) were purchased from Cell Signaling Tech (Danvers, MA). HRP-conjugated secondary antibodies and luminol reagents were purchased from Santa Cruz Biotech (Dallas, TX).
4
Assay for Nuclear Receptor Activation
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GW4064, (Z)-guggulsterone (GS), T0901317, 1α,25-vitamin D3, GW7647, and GW1929 were obtained from Cayman Chemical (Ann Arbor, MI, USA). CDCA, lithocholic acid, and Fast-blue BB salt were purchased from Sigma (St. Louis, MO, USA). GW501516 was from ChemScene LLC (Newark, NJ, USA). All-trans retinoic acid and 9-cis-retinoic acid were from FUJIFILM Wako Pure Chemical (Osaka, Japan). BMP-2 was from Miltenyi Biotec. (Bergisch Gladbach, Germany). Naphthol AS-MX phosphate was from Nacalai Tesque (Kyoto, Japan). Amino acid derivative and the coupling reagents were from Watanabe Chemical (Hiroshima, Japan) or Peptide Institute (Osaka, Japan). All other chemicals were from Tokyo Chemical Industry (Tokyo, Japan), FUJIFILM Wako Pure Chemical, and Aurora Fine Chemicals (San Diego, CA, USA) unless otherwise stated.
5
Assessing Macrophage-derived Organoid Viability
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The mouse macrophage cell line RAW 264.7 (ATCC TIB-71) was acquired and cultured according to the supplier’s instructions. FexD, OCA, and GW4064 were dissolved in DMSO. FexD was used as a custom production (Wuxi Biologics), and OCA and GW4064 were purchased from Cayman Chemicals. Organoids were treated with drugs on day 2 or day 3 after plating to capture the early growth phase. Images of organoid morphology changes after drug treatment were taken with EVOS M5000 microscope (Thermo Fisher Scientific). CellTiter-Glo Luminescent 3D Cell Viability Assay Kit (Promega, catalog G9683) was used to check the cell viability after drug treatment. Organoids were directly lysed using TRIzol reagent (Ambion, catalog 15596026), and RNA was extracted with RNeasy Mini Kit (QIAGEN, catalog 74106).
6
Evaluating Primary Hepatocyte Response
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Methylcellulose was obtained from Sigma-Aldrich (catalog no. 0262), GW4064 was obtained from Cayman Chemicals and 17OHP (Sigma-Aldrich).
Primary hepatocytes isolated from C57BL/6J mice were incubated with 100 μM DCA (Sigma-Aldrich) for 6 hours (n = 2 mice per group, three technical replicates).
Primary hepatocytes isolated from C57BL/6J mice were incubated with 100 μM DCA (Sigma-Aldrich) for 6 hours (n = 2 mice per group, three technical replicates).
7
Molecular Regulation of Phagocytosis
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Transcriptional activation of RAR‐reporter constructs by tRA was found to be maximal between 0.1 and 10 μM (Allenby et al. 1993 ), and experiments with glial cultures showed strong effects with 0.01–1 μM (van Neerven, Regen, et al., 2010 ). To test the molecular regulation of phagocytosis we therefore tested 10 nM, 0.1 μM, and 0.5 μM of the pan‐RAR agonist all‐tRA (R2625; Sigma); 0.1 and 0.5 μM pan‐RXR agonist bexarotene (153559‐49‐0; LC Laboratories); 1 μM pan‐RXR antagonist UVI3003 (3303; Tocris); 1 μM RARβ agonist‐RARα/γ antagonist BMS189453 (SML1149; identical to BMS453; Sigma); 1 and 10 μM pan LXR agonist T0901317 (Cay71810‐10; Cayman); 9 and 18 μM PPARα agonist fenofibrate (Cay10005368‐1; Cayman); 50 nM and 0.1 μM PPARγ antagonist rosiglitazone (LKT‐R5773.100; LKT Laboratories); 0.2 and 0.5 μM PPARβ/δ agonist GW501516 (Cay10004272‐1; Cayman); 1 and 2 μM FXR agonist GW4064 (Cay10006611‐5; Cayman); 0.5 and 1 μM TGM2 inhibitor cystamine dihydrochloride (B22873.14; Alfa Aesar); 15, 25, 50, and 100 μM TGM2 inhibitor ERW1041E (5095220001; Merck); and 1, 5, and 25 μM blocker of scavenger receptor CD36 sulfo‐N‐succinimidyl oleate (SSO; SML2148; Merck).
8
Inflammatory Pathways and Bile Acid Modulation
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Inflammatory pathways were induced with Escherichia coli LPS (isotype O26:B6; Sigma‐Aldrich) at concentrations of 0.1, 1, 10 and 100 ng/ml and with mouse IFNγ (Peprotech) 10 and 20 ng/ml. TUDCA sodium salt was purchased from Calbiochem (580549), sodium TLCA from Sigma‐Aldrich (T7515) and HDCA from Alfa Aesar (B20506). In preliminary experiments with Raw264.7 cells, using nitrite release as the inflammatory read out, we tested concentrations of 20–320 µM bile acids, dissolved in cell culture medium. Based on these results we chose 40 µM TLCA, 200 µM TUDCA and 160 µM HDCA in phagocytosis and gene expression assays. The activation of NFκB was inhibited with 2 µM Bay11‐7082 (Sigma‐Aldrich B5556). We blocked CD36 with 25 μM sulfo‐N‐succinimidyl oleate (SSO; Merck SML2148) and PKA with 2 µM H89 dihydrochloride (Sigma‐Aldrich B1427). All‐trans retinoic acid (RA; Sigma‐Aldrich R2625) was used at 0.1 µM and GW4064 (Cayman 10006611‐5) at 2 µM (Wu et al., 2021 (link)).
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