Male db/db (C57BL/KsJ) mice (8-wk old) and their age-matched wild-type (C57BL/6) mice were purchased from the Model Animal Research Center of Nanjing University. Mice were all housed in a temperature-controlled (20–24°C) and humidity-controlled (45–55%) room illuminated daily from 7am to 7pm (12-h light, 12-h dark cycle), with free access of water and standard laboratory chow. The mice were acclimated for 1 week prior to the start of experiments. db/db mice and WT mice (controls) were divided into two groups (10–15 per group), controls/vehicle [dimethylsulfoxide (DMSO)-treated] and TO901317-treated, respectively. TO901317 (TO), an LXR agonist (Cayman Chemical Co., Ann Arbor, MI. 30 mg/kg/d, dissolved in DMSO) or an equal volume of DMSO (control/vehicle) was given via intraperitoneal (IP) injection daily for 2 weeks. Food intake and body weight were measured everyday from the initiation of the treatment to the last day of experiments. To sacrifice the animals, mice were first anesthetized with isoflurane followed by cervical dislocation. All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai [SYXK (Shanghai 2009–0069)], and were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals [SCXK (Shanghai 2007–0005)].
To901317
Manufactured by Cayman Chemical
Sourced in United States
TO901317 is a synthetic compound that functions as a liver X receptor (LXR) agonist. It is commonly used in research applications to study the regulation of cholesterol, fatty acid, and glucose metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Dextran sulfate, by Merck Group (1 mentions)
Rpmi 1640, by Cayman Chemical (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
3h cholesterol, by American Radiolabeled Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Vinblastine, by Bio-Techne (1 mentions)
Ginkgolide a, by Santa Cruz Biotechnology (1 mentions)
Ginkgolideb, by Santa Cruz Biotechnology (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Torin 2, by Bio-Techne (1 mentions)
Desipramine, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Acldl, by Yeasen (1 mentions)
Oil red o, by Merck Group (1 mentions)
Cli 095, by InvivoGen (1 mentions)
Hpi 1, by Cayman Chemical (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Mplas, by InvivoGen (1 mentions)
Tgf β, by R&D Systems (1 mentions)
8 protocols using to901317
1
Investigating LXR Agonist Effects in Diabetic Mice
2
Cholesterol Efflux Assay Protocol
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Cholesterol efflux assay was performed as previously described [27 (link)]. Briefly, THP-1 monocytes were differentiated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Merck) and incubated in serum-containing RPMI-1640 (Thermo Fisher Scientific, USA) with 0.6 μCi/ml [3H]-cholesterol (American Radiolabeled Chemicals Inc., USA) for 72 h. Cells were then incubated for 18 h in serum-free RPMI-1640 containing 4 μM LXR agonist TO-901317 (Cayman Chemical, USA). Cholesterol efflux was performed using 1.1% apoB-depleted plasma for 2 h. ApoB-depleted plasma was obtained after precipitation of apoB-containing lipoproteins with Dextran Sulfate (Merck, USA) as previously described [26 (link)].
3
Quantifying Ligand Binding to PXR
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Vinblastine was obtained from Tocris Bioscience
(Minneapolis, MN, USA); GST-hPXR-LBD, LanthaScreen Tb-anti-GST antibody,
TR-FRET PXR (SXR) assay buffer, BODIPY FL Vinblastine, and 1 M DTT
(dithiothreitol) were purchased from Invitrogen (Carlsbad, CA, USA);
human glutathione S transferase protein (GST) was purchased from Abcam
(Cambridge, MA, USA); catharanthine and vindoline were purchased from
LKT Laboratories, Inc. (St. Paul, MN, USA); deacetyl Vinblastine was
purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario,
Canada); dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific
(Pittsburgh, PA, USA); TO901317 was purchased from Cayman Chemical
(Ann Arbor, MI, USA); SR12813 was purchased from Enzo Life Sciences
(Farmingdale, NY, USA); clotrimazole, rifampicin, 2-amino-2-(hydroxymethyl)-1,3-propanediol
(Tris), potassium chloride (KCl), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
hydrate (CHAPS), and bovine serum albumin (BSA) were purchased from
Sigma (St. Louis, MO, USA); hyperforin, ginkgolide A, and ginkgolide
B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA);
and black 384-well polypropylene plates were purchased from Matrical
Bioscience (Spokane, WA, USA).
(Minneapolis, MN, USA); GST-hPXR-LBD, LanthaScreen Tb-anti-GST antibody,
TR-FRET PXR (SXR) assay buffer, BODIPY FL Vinblastine, and 1 M DTT
(dithiothreitol) were purchased from Invitrogen (Carlsbad, CA, USA);
human glutathione S transferase protein (GST) was purchased from Abcam
(Cambridge, MA, USA); catharanthine and vindoline were purchased from
LKT Laboratories, Inc. (St. Paul, MN, USA); deacetyl Vinblastine was
purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario,
Canada); dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific
(Pittsburgh, PA, USA); TO901317 was purchased from Cayman Chemical
(Ann Arbor, MI, USA); SR12813 was purchased from Enzo Life Sciences
(Farmingdale, NY, USA); clotrimazole, rifampicin, 2-amino-2-(hydroxymethyl)-1,3-propanediol
(Tris), potassium chloride (KCl), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
hydrate (CHAPS), and bovine serum albumin (BSA) were purchased from
Sigma (St. Louis, MO, USA); hyperforin, ginkgolide A, and ginkgolide
B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA);
and black 384-well polypropylene plates were purchased from Matrical
Bioscience (Spokane, WA, USA).
4
Modulation of A2E-induced RPE Responses
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The lipofuscin bisretinoid A2E was synthesized according to published protocols and purified by high-performance liquid chromatography (HPLC; >97%, electrospray ionization mass spectrometry; Lakkaraju et al., 2007 (link)). RPE were exposed to either a chronic low-dose of A2E (50 nM for 3 wk) or an acute high-dose of A2E (10 μM for 6 h, followed by a 48-h chase). Quantification of A2E levels in cells was performed by HPLC as previously reported (Radu et al., 2011 (link)). Other drugs used were the mTOR inhibitors Torin 1 and Torin 2 (50 nM and 1.5 μM, respectively, for 2 h; Tocris Bioscience, Bristol, UK), the vacuolar ATPase inhibitor bafilomycin A1 (100 nM for 2 h; EMD Millipore, Billerica, MA), the LXRα agonist TO901317 (1 μM for 20 h; Cayman Chemicals, Ann Arbor, MI), the HDAC6 inhibitor TSA (500 nM, 16 h; Sigma-Aldrich), and the ASMase inhibitor desipramine (10 μM for 3 h; Sigma-Aldrich). For depolymerization of MTs, cells were treated with 33 μM nocodazole for 30 min; this was followed by cold treatment (4°C) for 30 min (Kreitzer et al., 2003 (link)). At the concentrations and exposure times used, none of these drugs caused alterations in RPE cell morphology or physiology (monitored by TER measurements and ZO-1 and organelle marker staining).
5
Lipid Accumulation Assay in Fibroblasts
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Fibroblasts were cultured from ear skin tissues of the ABCA12+/+, ABCA12+/Z9, and ABCA12Z9/Z9 pigs. Assays of lipid accumulation were performed according to instruction previously described (Smyth et al., 2008 ). Fibroblasts were incubated in serum-containing medium with TO-901317 (4 mM; Cayman Chemical) and AcLDL (10 mg/ml; Yeasen Biotech) for 18 hrs. Fibroblasts were washed with PBS, fixed with 4% formaldehyde, and then stained with Oil Red O (Sigma-Aldrich) working solution.
6
Differentiation and Activation of Macrophages
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RAW264.7 mouse macrophages and THP-1 human monocytes were purchased from American Tissue Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM or RPMI containing 10% fetal bovine serum (FBS) and antibiotics, respectively, as previously reported. THP-1 culture medium also contained 0.05 mM β-mercaptoethanol. THP-1 differentiation into macrophages was achieved by treating the cells in their culture medium with 40 nM phorbol 12-myristate 13-acetate (PMA) for 48 h, detached, PMA removed, and cells plated in 12 well plates for experiments. Differentiation into macrophages was confirmed by CD14 mRNA expression. For experiments, when cells were at approximately 80% confluence, FBS concentration was reduced to 0.1% at the time treatments were initiated and for the 48 h duration of the studies. LPS and PMA were purchased from Sigma-Aldrich (St. Louis, MO, USA), TGF-β from R&D Systems (Minneapolis, MN), MPLAs, CLI-095, and Poly(I:C) from InvivoGen (San Diego, CA, USA), and CU-T12-9, HPI-1, HPI-4, and TO901317 from Cayman Chemical (Ann Arbor, MI, USA). All oxysterols and sterols were purchased from Sigma. Oxysterols were prepared in-house according to our published protocols and those outlined in the present manuscript [34 (link),36 (link)].
7
Abcd1 Knockout Mouse Model Protocol
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The mouse work involved adult, male Abcd1+/y (WT) or Abcd1−/y (KO) mice with the Abcd1tm1Kan/J allele [34 (link)] back-crossed to the C57BL/6J strain for over 20 generations. All mice for the study were bred and maintained at the in-house facility of the Medical University of Vienna in a temperature and humidity-controlled room on a 12:12 h light–dark cycle with free access to water and standard rodent chow (R/M-H, Ssniff®, Soest, Germany). Alternatively, at the age of 6.5 months, mice received R/M-H chow supplemented with 100 mg/kg TO901317 (Cayman Chemical, pressed into pellets by Ssniff) for 10 weeks [32 (link)]. Experimental cohorts were generated by mating heterozygous females and C57BL/6J males producing Abcd1 KO and WT male littermates, for which the genotype was obtained by PCR as described previously [35 (link)], and sacrificed for brain and spinal cord analysis at the age of 8 months (lipidomics) or 12 months (RT-qPCR and histology). All procedures were in compliance with Austrian regulations (BGBl. II Nr. 522/2012) and the European Union Directive 2010/63/EU for humane care and handling. The study procedures were approved by the local Animal Care and Use Committee of the Medical University of Vienna and by the Austrian Federal Ministry of Education, Science and Research (BMBWF-66.009/0174-V/3b/2019).
8
Evaluating LXRα Agonist Activity
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EPA, polyoxyethylene (20) sorbitan monolaurate (Tween 20) , and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) . A synthetic LXRα agonist, TO-901317, was purchased from Cayman Chemicals (Ann Arbor, MI, USA) .
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