Gw3965

Manufactured by Cayman Chemical

Sourced in United States

GW3965 is a synthetic ligand for the liver X receptor (LXR) that can be used in research applications. It functions as an agonist to activate LXR, a transcription factor involved in the regulation of cholesterol and lipid homeostasis.

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Lab products found in correlation

19 protocols using gw3965

Regulation of Choline-Containing Phospholipid Release in Fibroblast-Like Synoviocytes

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To study the release of choline-containing PLs from FLS during treatment, confluent cells from Passage 5 were cultured in 6-well plates and labeled with 5 µCi/mL [³H]-choline chloride (PerkinElmer, Rodgau, Germany) for 24 h in choline-depleted, phenol red-free DMEM (PAN Biotech) that was supplemented as above. FLS were washed thoroughly to remove unincorporated precursors and starved for 24 h with supplemented DMEM that contained 5% FBS. After a change in media, cells were treated for 48 h with 0.1 µM T0901317 (Sigma-Aldrich, Taufkirchen, Germany) or GW3965 (Cayman Chemical Company, Ann Arbor, MI, USA); 1 ng/mL IL-1ß (PeproTech, Hamburg, Germany), and 1 µM of the P38 MAPK inhibitor SB203580, 0.1 µM of the NF-κB inhibitor QNZ, the broad-spectrum JNK inhibitor SP600125 (Selleck Chemicals, Munich, Germany), or a negative control. The media and treatment were changed after 24 h. The release of radiolabeled PLs was measured in media from the final 24 h of the experiment. The experiment was repeated 4–8 times using FLS from 5 to 9 OA patients (age 53–81 years (69.3 ± 8.2), BMI 26.8–34.9 kg/m² (30.1 ± 2.4), Kellgren-Lawrence scores of 2–4 (3.1 ± 0.8), CRP 3.18 ± 4.51 mg/L, 3 males, 6 females).

Thottakkattumana Parameswaran V., Hild C., Eichner G., Ishaque B., Rickert M, & Steinmeyer J. (2022). Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-Like Synoviocytes. International Journal of Molecular Sciences, 23(5), 2409.

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Investigating Cell Signaling Pathways

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GW3965 (for cell‐based assays), propidium iodide, and MG132 were provided from Sigma‐Aldrich (St Louis, MO). Transforming growth factor‐beta 1 (TGF‐β1) was purchased from Humanzyme (Chicago, IL), whereas fluorescein isothiocyanate–annexin V was from BD Biosciences (San Jose, CA). GW3965 (for the in vivo experiment), SR144528, and SB525334 were supplied from Cayman Chemical (Ann Arbor, MI). JWH133 was provided from Tocris Bioscience (Bristol, United Kingdom). Clodronate liposome was purchased from FormMax (Sunnyvale, CA). mTNF‐α was supplied from R&D Systems (Minneapolis, MN), and horseradish peroxidase–conjugated goat antirabbit and goat antimouse immunoglobulin Gs were from Zymed Laboratories (San Francisco, CA). Antibody information is provided in Supporting Table S1.

Wu H.M., Kim T.H., Kim A., Koo J.H., Joo M.S, & Kim S.G. (2019). Liver X Receptor α–Induced Cannabinoid Receptor 2 Inhibits Ubiquitin‐Specific Peptidase 4 Through miR‐27b, Protecting Hepatocytes From TGF‐β. Hepatology Communications, 3(10), 1373-1387.

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Synthesis and Characterization of Oxysterol Compounds

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27HC (purity >95%) and dendrogenin A (purity >98%) were synthesized by Sai Life (Hyderabad, India). 25HC, 4βHC and Desmosterol were synthesized by Steraloids (Newport, RI) (purity >98%). 22(R)HC was synthesized by Avanti (Alabaster, AL) (purity >99%). 24(S)HC and 24,25-epoxycholesterol (24,25EC) were synthesized by Enzo (Farmingdale, NY) (purity ≥98%). GW3965, T0901317, GSK2033 and 7-ketocholesterol (7KC) were synthesized by Cayman (Ann Arbor, MI) (purity ≥98%). 20(S)HC was synthesized by Tocris (Bristol, UK) (purity >95%). 17β-Estradiol (E2) was synthesized by MP Biomedicals (Irvine, California) (purity ≥98%). 27HC, dendrogenin A, 24(S)HC, 20(S)HC, GW3965, T0901317 and GSK2033 were dissolved in dimethyl sulfoxide (DMSO) and stored in −20°C. 7KC, 4βHC, 22(R)HC, 24,25EC and 25HC were dissolved in ethanol and stored in −20°C. Desmosterol was dissolved in M-pyrol and stored in −20°C. Antibodies for flow cytometry were purchased from BD Biosciences and used at a working dilution of 1:100 in FACS buffer (2% fetal bovine serum (FBS) in phosphate buffered saline (PBS)). Catalogue numbers were as follows: CD3: 555275, CD4: 553729, CD8: 557682. Annexin V-FITC/propidium iodide apoptosis assay was purchased from Thermo Fisher (catalogue number: V13242), and FAM-DEVD-FMK/propidium iodide apoptosis assay was purchased from Immunochemistry (catalogue number: 94).

Ma L., Wang L., Nelson A.T., Han C., He S., Henn M.A., Menon K., Chen J.J., Baek A.E., Vardanyan A., Shahoei S.H., Park S., Shapiro D.J., Nanjappa S.G, & Nelson E.R. (2020). 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression.. Cancer letters, 493, 266-283.

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Chronic Asthma Model in LXR KO Mice

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Mice were randomly divided into six groups (n=6 mice/group) as follows: Control group, ovalbumin (OVA) group, GW3965 group, LXRα^-/- group, LXRβ^-/- group and LXRα/β knockout (KO) group. Certainwild-type mice received intervention with the LXR agonist GW3965 (Cayman Chemical Company) at 20 mg/kg by intraperitoneal injection. LXR-KOmice and wild-type mice were used to establish a chronic asthma model (18 (link)). Mice were intraperitoneally injected with 0.1 ml sensitizing solution [0.1 mg OVA; 0.07 ml Imject™ Alum Adjuvant (Beijing Biolead Biotechnology Co., Ltd.); 0.03 ml PBS] on days 0, 7 and 14. The mice were stimulated with 1% OVA solution using an ultrasonicnebulizer (402AI; Jiangsu Yuyue Medical Equipment & Supply Co., Ltd.) from day 21 for 30 min each time, three times a week, for 8 consecutive weeks. Mice treated with the LXR agonist were administered an intraperitoneal injection of GW3965 at 20 mg/kg (10 (link),22 (link)) prior to each stimulation.

Zhang J., Wu Z., Yu F., Ye L., Gu W., Tan Y., Wang L, & Shi Y. (2021). Role of liver-X-receptors in airway remodeling in mice with chronic allergic asthma. Experimental and Therapeutic Medicine, 22(3), 920.

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Cholesterol Metabolism Pathway Assays

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Antibodies for flow cytometry were purchased from eBioscience. Antibodies for Western blot against Srebp1, Srebp2, LXRs, and Sumo1, -2, and -3 were purchased from Abcam, and other Western blot antibodies were purchased from Cell Signaling Technology. Cytokines were purchased from R&D Systems, and neutralization antibodies were purchased from Bio X Cell unless otherwise indicated. Cholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 7β-hydroxycholesterol, 20α-hydroxycholesterol, 3β-Hydroxy-5-cholesten-7-one, 22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 5β-Cholestan-3β-ol, 5α-Cholest-7-en-3β-ol, 5,24-Cholestadien-3β-ol, 5-α-cholestane, β-cyclodextrin, simvastatin, lovastatin, and 2-D08 were purchased from Sigma-Aldrich. T0 and GW3965 were purchased from Cayman Chemical. Overexpression plasmids were purchased from Addgene unless otherwise indicated. All experiments were conducted according to the manufacturer’s protocols unless otherwise indicated.

Ma X., Bi E., Huang C., Lu Y., Xue G., Guo X., Wang A., Yang M., Qian J., Dong C, & Yi Q. (2018). Cholesterol negatively regulates IL-9–producing CD8+ T cell differentiation and antitumor activity. The Journal of Experimental Medicine, 215(6), 1555-1569.

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Investigating Macrophage Immune Response to MHV68 Infection

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Wild-type MHV68 viral stocks were prepared and titers determined on NIH 3T12 cells. Bone marrow-derived macrophages or MEFs were infected with live or UV-inactivated MHV68 at the indicated multiplicity of infection (MOI) for 1 h to allow adsorption and were washed 2 to 3 times with phosphate-buffered saline (PBS) prior to medium replenishment. UV inactivation of MHV68 was performed as previously described (17 (link)). Supplementation experiments were performed by adding the indicated compound(s) to the replenishment media following viral adsorption. GW3965 and TOFA [tetradecyloxy-2-furoic acid] were purchased from Cayman Chemical (Ann Arbor, MI). GW3965 was dissolved in dimethyl sulfoxide (DMSO) and then diluted in cell culture media to a concentration of 5 µM. TOFA was dissolved in DMSO and then diluted in cell culture media to a concentration of 10 µg/ml. Control cell cultures were treated with equivalent amounts of DMSO.

Lange P.T., Schorl C., Sahoo D, & Tarakanova V.L. (2018). Liver X Receptors Suppress Activity of Cholesterol and Fatty Acid Synthesis Pathways To Oppose Gammaherpesvirus Replication. mBio, 9(4), e01115-18.

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LXR Agonist and Inhibitor Screening

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Novel LXR ligand GAC0001E5 (1E5) was synthesized by Otavachemicals (Concord, ON, Canada). LXR agonist GW3965 was received from Cayman Chemicals (Ann Arbor, MI, USA #10054). Tyrosine kinase inhibitor lapatinib and fatty acid synthase inhibitor C75 were purchased from MedChemExpress [South Brunswick Township, NJ, USA (lapatinib #HY-50898, C75 #HY-12364)]. AU565, HCC-1954, and SKBR3 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). AU565 and HCC-1954 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA #11875085). SKBR3 cells were cultured in DMEM (Gibco, #12430047) containing high glucose and HEPES. All media were supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, #26140079). All cell cultures were maintained in a humidified incubator of 5% CO₂ at 37 °C.

Premaratne A., Basu S., Bagchi A., Zhou T., Feng Q, & Lin C.Y. (2024). Liver X Receptor Ligand GAC0001E5 Downregulates Antioxidant Capacity and ERBB2/HER2 Expression in HER2-Positive Breast Cancer Cells. Cancers, 16(9), 1651.

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HDM-Induced Asthma Model in Mice

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Mice were sensitized to HDM by intratracheal (i.t.) instillation of 1 μg HDM extracts (Greer Laboratories, Lenoir, NC) on days 0 and 14. Starting from day 21, mice were challenged intranasally with 10 μg HDM for 4 consecutive days. Mice that were sensitized and challenged with PBS served as controls. In the LXR agonist studies, mice were treated with 20 mg/kg GW3965 (Cayman Chemicals, Ann Arbor, MI) by oral gavage 2 h before intranasal HDM challenge.

Smet M., Van Hoecke L., De Beuckelaer A., Vander Beken S., Naessens T., Vergote K., Willart M., Lambrecht B.N., Gustafsson J., Steffensen K.R, & Grooten J. (2016). Cholesterol‐sensing liver X receptors stimulate Th2‐driven allergic eosinophilic asthma in mice. Immunity, Inflammation and Disease, 4(3), 350-361.

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Agonist-Induced APOF mRNA Expression

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C3A cells, derived from the human hepatocellular carcinoma HepG2 cell line, were purchased from American Type Culture Collection (ATCC, Manassas VA). Cells were grown to near confluence in Eagle’s minimum essential media (MEM) containing 10% fetal calf serum. For experiments, cells were washed with MEM then incubated with the indicated agonist diluted in either MEM containing 5% lipoprotein-deficient human serum or in Opti-MEM (Life Technologies, Grand Island, NY). After the indicated time, cells were washed with PBS and total RNA was extracted. There was no consistent difference in the response of APOF mRNA levels to agonist between these two media conditions. 22(R)- and 22(S)-hydroxycholesterol, chenodeoxycholate, 9-cis retinoic acid, and actinomycin D were purchased from Sigma Aldrich (St. Louis, MO). Clofibrate, GW3965, and GW7647 were from Cayman Chemicals (Ann Arbor, MI). Stock solutions of nuclear receptor agonists were prepared in DMSO.

Liu Y., Izem L, & Morton R.E. (2019). Identification of a hormone response element that mediates suppression of APOF by LXR and PPARα agonists. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1865(3), 158583.

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Synthesis and Characterization of Novel LXR Ligand

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Novel LXR ligand GAC0001E5 was synthesized by Otavachemicals, Concord, ON, Canada. Synthetic LXR agonist GW3965 and glutaminase inhibitor BPTES were obtained from Cayman Chemical, Ann Arbor, MI, USA. All cell culture media and FBS were purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA. MCF-7 and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. A Tamoxifen-resistant MCF-7-TamR cell line was obtained from the Bawa-Khalfe Lab. MCF-7 cells were treated with 1 μM of 4-Hydroxytamoxifen (Selleckchem, Houston, TX, USA) for six months for acquisition of resistance to produce MCF-7-TamR. Furthermore, they were supplemented with 1 μM of 4-Hydroxytamoxifen to maintain resistance [18 (link)]. MCF-7 and MCF-7-TamR cell lines were cultured in DMEM media (Gibco #12430054). MDA-MB-231 cells were cultured in DMEM/F12 (Ham) (Gibco #11330032). Each media type was supplemented with 10% FBS (Gibco #26140079). Cell cultures were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

Premaratne A., Ho C., Basu S., Khan A.F., Bawa-Khalfe T, & Lin C.Y. (2023). Liver X Receptor Inverse Agonist GAC0001E5 Impedes Glutaminolysis and Disrupts Redox Homeostasis in Breast Cancer Cells. Biomolecules, 13(2), 345.

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