P2-CDCs (10,000/cm2) were seeded on either FN or ColIV coated plates containing cardiomyogenic differentiation medium-1 (CDM-1; Table S1 ) and were treated with 5-Azacytidine (5-Aza) for 3 days, followed by ascorbic acid (AA; Sigma, UK) from day 6–14 (Fig. 2 A). For later experiments, P2-CDCs (20,000/cm2) created through CoIV/HD culture, were seeded in CEM on plates coated with 1 mg/ml FN in 0.01% gelatin. Once cells reached confluency, cardiomyogenic differentiation medium-2 (CDM-2 Table S1 ) containing 5-Aza (5 µM) was added for 3 days. On day 4, the medium was changed for CDM-2 only without 5-Aza and refreshed every 2–3 days until day 7. On day 7 medium was replaced with one of the following media: TGFβ: CDM-2 containing 1 ng/ml TGF-β (Peprotech, UK) and 0.1 mM AA; or WY-14643: CDM-2 containing 1 ng/ml TGF-β and 0.1 mM AA plus 100 µM WY-14643 (PPARα agonist, Sigma, UK), each alternating with CDM-2 containing 0.1 mM AA until day 30 as shown in Fig. 5 A. Cytotoxicity testing for 5-Aza and WY-14643 is shown in Fig. S3 .
Wy14643
Manufactured by Merck Group
Sourced in United States, China, Germany, United Kingdom
WY14643 is a laboratory chemical product manufactured by Merck Group. It is a peroxisome proliferator-activated receptor alpha (PPARα) agonist. The core function of WY14643 is to serve as a research tool for studies related to PPARα activation and its effects in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Fenofibrate, by Merck Group (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Gw6471, by Merck Group (5 mentions)
Gw501516, by Merck Group (5 mentions)
Rosiglitazone, by Merck Group (5 mentions)
Gemfibrozil, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (4 mentions)
Gw9662, by Merck Group (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Oleic acid, by Merck Group (3 mentions)
Gw0742, by Bio-Techne (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Hexadecanamide, by Merck Group (2 mentions)
Clofibrate, by Merck Group (2 mentions)
9 octadecenamide, by Merck Group (2 mentions)
Mtt based toxicity assay kit stock no tox 1, by Merck Group (2 mentions)
61 protocols using wy14643
1
Cardiomyogenic Differentiation of CDCs
2
Fasting and WY-14,643 Treatment in Mice
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Mice were housed under specific pathogen free (SPF) conditions, at 22 °C, and with 12 hours dark/light cycles (light cycle from 8 am to 8 pm). Mice were fed with standard chow diet (Harlan Teklad 2018, 18% of fat-based caloric content) or fasted for 48 or 24 hours, with ad libitum access to water, when specified. Complete necropsies were performed in parallel when ad libitum and fasted mice were compared. All the mice used in this work are C57BL6J/Ola.Hsd males of 10–16 weeks of age. Temperature was measured using a rectal thermometer (Braintree Scientific). For administration of WY-14,643, mice were fasted overnight, orally treated by gavage with 75 mg/kg WY-14,643 (Sigma) dissolved in PEG300 (Sigma) and 10% N-methyl-2-pyrrolidone (Sigma) and sacrificed 6 hours later. When combined with 48 hours fasting, mice were treated four times with 75 mg/kg WY-14,643 by oral gavage (at the beginning of fasting and after 8, 24 and 32 hours of fasting).
3
Evaluating Cellular Responses to Molecular Compounds
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Seventeen kinds of amino acid were purchased from Shanghai Anpel (Shanghai, China). Gallic acid, glucose, gamma-aminobutyric acid, and VB3 were purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), DF-12 medium, fetal bovine serum (FBS), calf serum (CS), phosphate-buffered saline (PBS), and penicillin–streptomycin were purchased from Gibco (Carlsbad, CA, USA). EpiGrowth medium was purchased from Guangdong Biocell (Guangzhou, China). A CCK-8 Test Kit was purchased from Dojindo (Kumamoto, Japan). DMSO, WY14643 and VE were purchased from Sigma (Saint Louis, MO, USA). WY14643 and VE were purchased from Sigma (Saint Louis, MO, USA). RNAiso Plus, a reverse transcription kit, and fluorescent dye were purchased from Accurate Biology Co., Ltd. (Changsha, China). ROS kit and a DCFH-DA probe were purchased from Beyotime (Shanghai, China). TGF-β1 was purchased from Peprotech (Rocky Hill, NJ, USA). Primary antibody and secondary antibody were purchased from Abcam (Cambridge, UK).
4
Seaweed Extracts Modulate Nuclear Receptor Activity
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Transfected cells were seeded in a 96-well luminescence plate. After 24 h, the cells were incubated for 24 h in phenol red-free DMEM/F-12 medium (ThermoFisher Scientific) with the seaweed extracts (dosages based on saringosterol content), the LXRα/β agonist T0901317 (1 µM; #293754-55-9; Cayman, Ann Arbor, MI, USA), PPARα agonist WY-14643 (50 µM; #50892-23-4; Merck), PPARγ agonist pioglitazone (10 µM; #111025-46-8; Cayman), or the extract/compound solvents ethanol or DMSO. Cells were lysed in 25 μL lysis buffer and the Firefly and Renilla luminescent signals were measured using the Dual-Luciferase® Reporter assay system (Promega) and a Victor X4 plate reader (PerkinElmer, Groningen, The Netherlands). The relative receptor activity was defined as the ratio of Firefly luminescence to Renilla luminescence. The fold change was defined as the ratio of the relative receptor activity of seaweed- or agonist-exposed cells to the relative receptor activity of ethanol-exposed cells. The experiments, with the stimulation performed in triplicate, were repeated ≥ three times.
5
Inflammasome Activation and PPARα Agonism
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PYRIN inflammasome activation was obtained by addition of 10 ng/mL ultra-pure E.coli LPS (tlrl-3pelps, InvivoGen) and 1 µg/mL C3 toxin (CT04-B, Cytoskeleton) in serum reduced media, Optimem (31985062, ThermoFisher Scientific) for 16 hours. Monocytes with 10 ng/mL LPS addition or no stimuli for 16 hours in Optimem were included as LPS-and baseline controls, respectively. For PPARα activation, PPARα agonist WY14643 (C7081, Merck) or vehicle (DMSO) was added at 50 ng/mL for 16 hours together with LPS (10ng/mL) and C3 (1 µg/mL). Cell supernatant was removed, cleared by centrifugation and stored at -30°C until protein quantifications. Cells were lyzed by addition of TRI Reagent (AM9738, ThermoFisher Scientific) and cell lysate was stored at -80°C until RNA extraction.
6
Phytochemical Analysis of Natural Antioxidants
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Sigma-Aldrich (St. Louis, MO, USA) provided 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2′,7′-dichlorofluorescein diacetate (DCF-DA), lipopolysaccharide (LPS), Folin–Ciocalteu (F–C) reagent, gallic acid, AlCl3, Griess reagent, NG-Methyl-L-arginine acetate salt (L-NMMA), WY14643, CaCl2, and HPLC grade standards of quercetin, kaempferol, isorhamnetin, kaempferol 3-glucoside, isorhamnetin 3-glucoside, quercetin 3-glucoside, and quercetin 3,4′-diglucoside. Solvents for extraction and fractionation (ethanol, n-hexane, chloroform, ethyl acetate, and n-butanol) and formic acid were purchased from Daejung Chemicals & Metals (Siheung, Republic of Korea). Solvents for LC/MS (methanol, acetonitrile) were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA).
7
Collagen IV Immunofluorescence Assay
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Collagen IV from human placenta (C5533), PBS (D8537), Triton-X 100 (T8787), DMEM (D5796), DAPI (D8417), β-mercapto-ethanol (M6250), fluorescein sodium (F6377), N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (S6510), Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin (B4636), Z-Leu-Leu-Glu-7-amido-4-methylcoumarin (C0483), MG132 (C2211, Z-Leu-Leu-Leu-al), Lactacystin (L6785), WY-14643 (C7081), GW6471 (G5045), rhPAI-1 (A8111), albumin from bovine serum for immunofluorescence microscopy (fraction V, A9647) and for western blotting (A7906) were purchased from Sigma–Aldrich. DMEM without glucose (11966-025, Gibco®) was obtained from Life technologies (USA). FCS Gold EU approved was bought from PAA Laboratories (A15151, Lot A15111-2018, Linz, Austria) and was heat-inactivated in a water-bath at 56°C for 30 min. Penicillin/streptomycin (100X, 10,000 Units/mL, 10,000 μg/mL, A2213) and 0.05% Tyrpsin/0.02% EDTA-solution (L2143) were from BioChrom AG (Berlin, Germany). 6-well and 24-well plates and 24-well Transwell® inserts (0.4 μm pore size, PET) were obtained from Becton and Dickinson (REF353046, REF353035, REF353226, USA). Gelatine was from SERVA (22151, Heidelberg, Germany), nuclease-free water was purchased from Ambion (AM9937, USA). All other substances were of analytical grade.
8
Apoptosis Signaling Pathway Analysis
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Doxorubicin, Cycloheximide (CHX), MG132, WY-14643, and Annexin V-FITC Apoptosis Detection Kit were purchased from Sigma-Aldrich (MA, USA). Antibodies against Flag, Caspase-3, Caspase-8, Caspase-9, PARP, Bax, phospho-Bad (Ser112), Cytochrome C, p53, p53(Ser15), p53(Ser20), p73, and GAPDH were purchased from Cell Signaling Technology (Boston, USA). Antibodies against ACOX1 was purchased from Abcam (Cambridge, U.K.). Antibody against puromycine was purchased from Merk (Darmstadt, Germany). LEHD, DEVD, and IETD were purchased from R&D Systems (MN, USA). Raji and Daudi cells were obtained from the ATCC (Manassas, VA, USA) and were cultured in RPMI-1640 (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
9
Investigating PPAR Receptors and Neuronal Markers
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Rabbit polyclonal anti-PPARα antibody (Abcam; Cat# ab189159; WB and IHC), mouse anti-NeuN antibody (Millipore; Cat# MAB377), rabbit polyclonal anti-PPARβ antibody (Abcam; Cat # ab8937; WB and IHC), anti-PPARγ antibody (Abcam; Cat# ab66343; WB and IHC), anti-NMDAR2A antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #4205; Abcam for IHC, Cat# ab169873), anti-GluR1 antibody (Cell Signaling for WB at a dilution of 1:1000, Cat #13185; Abcam for IHC, Cat # ab131507), anti-CREB antibody ( Cell Signaling for WB at a dilution of 1:1000 and IC at a dilution of 1:200, Cat# 9104), and anti-Arc antibody (Abcam for WB at a dilution of 1:1000, Cat # ab118929) were used in this study. Different pharmacological compounds including 9-octadecenamide (Cat#O2136), hexadecanamide (Cat#S350435), 2,4-bis(α,α-dimethyl benzyl) phenol (Cat #372129), gemfibrozil (Cat #G9518), clofibrate (Cat# C6643), fenofibrate (Cat# F6020), GW9662 (Cat# M6191), WY-14643 (Cat# C7081), and MTT-based toxicity assay kit (Stock No. TOX-1) were purchased from Sigma-Aldrich. GST-PPARα-LBD and GST-PPARβ-LBD were purchased from Protein One. On the other hand, 3-hydroxy 2, 2-dimethyl butyric acid ethyl ester (Cat# sc-216452) was purchased from Santa Cruz.
10
Acute Liver Failure Mouse Model
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Male C57BL/6 mice at the age of 8-12 weeks were purchased from the Capital Medical University (Beijing, China) and fed freely with a standard chow diet and water; they were housed under specific pathogen-free conditions for 1 week before the experiments. All animals received humane care according to the Capital Medical University Animal Care Committee guidelines.
The mice were intraperitoneally injected with D-GalN (700 mg/kg; Sigma, St. Louis, MO, USA) and LPS (10 μg/kg; InvivoGen, San Diego, CA, USA) to induce ALF, or with saline in the control animals. The PPARα activator Wy-14643 (6 mg/kg; Sigma) was administered via injection into the tail vein 2 h prior to D-GalN/LPS exposure. The downregulation of PPARα and CHOP were achieved by tail vein injection of specific siRNA (50 μM/kg; Jima, Suzhou, China). A chemical chaperone that relieves ER stress, 4-PBA (100 mg/kg; Sigma), was dissolved in PBS and administered intraperitoneally 6 h prior to D-GalN/LPS exposure. The mice were euthanized at 6 h after D-GalN/LPS treatment, and liver and serum samples were collected for future analysis.
The mice were intraperitoneally injected with D-GalN (700 mg/kg; Sigma, St. Louis, MO, USA) and LPS (10 μg/kg; InvivoGen, San Diego, CA, USA) to induce ALF, or with saline in the control animals. The PPARα activator Wy-14643 (6 mg/kg; Sigma) was administered via injection into the tail vein 2 h prior to D-GalN/LPS exposure. The downregulation of PPARα and CHOP were achieved by tail vein injection of specific siRNA (50 μM/kg; Jima, Suzhou, China). A chemical chaperone that relieves ER stress, 4-PBA (100 mg/kg; Sigma), was dissolved in PBS and administered intraperitoneally 6 h prior to D-GalN/LPS exposure. The mice were euthanized at 6 h after D-GalN/LPS treatment, and liver and serum samples were collected for future analysis.
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