FAO cells were plated in 96 well plates (black bottom) for 24h and maintained at 37 °C and 5% CO2. Cells were treated with (0.25 mM, 0.5 mM and 1mM) Clofibrate (Sigma) or DMSO (vehicle control) for 1h. Tert-butyl hydroperoxide (TBHP) served as a positive control in this experiment. Cells were stained with DCFDA for 30 minutes and followed by measurement of the absorbance using a fluorescent plate reader (Synergy H1 Hybrid, BioTek) with excitation wavelength at 485 nm and emission wavelength at 535 nm. For DHE staining, FAO cells were plated on chamber slides for 24 h and maintained at 37 °C and 5% CO2. Cells were treated with 0.25 mM Clofibrate (Sigma) for 1 h or DMSO (vehicle control). The cells were incubated with 5 μM DHE (in PBS) for 30 minutes at 37 °C in a dark chamber, fixed for 10 minutes in 4% paraformaldehyde and images promptly captured using an Olympus BX40 fluorescence microscope.
Clofibrate
Manufactured by Merck Group
Sourced in United States, United Kingdom
Clofibrate is a pharmaceutical compound used in laboratory settings. It functions as a lipid-regulating agent, playing a role in the control and management of lipid levels. This description is intended to be factual and unbiased, without any extrapolation on the intended use of the product.
Automatically generated - may contain errors
Lab products found in correlation
Palmitic acid, by Merck Group (3 mentions)
Gemfibrozil, by Merck Group (3 mentions)
Fenofibrate, by Merck Group (3 mentions)
Hexadecanamide, 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)
Gw9662, by Merck Group (2 mentions)
Wy14643, by Merck Group (2 mentions)
Bx40 fluorescence microscope, by Olympus (2 mentions)
Synergy h1 hybrid, by Agilent Technologies (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Bezafibrate, by Merck Group (2 mentions)
Gw6471, by Bio-Techne (2 mentions)
Etomoxir, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Dimethyl α ketoglutarate, by Merck Group (1 mentions)
Malonyl coa, by Merck Group (1 mentions)
Ab109362, by Abcam (1 mentions)
Deferiprone, by Merck Group (1 mentions)
4 pba, by Merck Group (1 mentions)
17 protocols using clofibrate
1
Clofibrate-induced Oxidative Stress Assay
2
Clofibrate-induced Oxidative Stress Assay
3
Reconstitution of Metabolic Compounds
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Clofibrate and palmitic acid (Sigma-Aldrich, Dorset, UK) were all reconstituted in dimethyl sulfoxide (DMSO). Etomoxir, malonyl-CoA, glucose, dimethyl-α-ketoglutarate, and glutamine (Sigma-Aldrich, Dorset, UK) were reconstituted in E199 medium.
4
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.
5
Mitochondrial and Autophagy Pathway Analysis
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Antibodies and other reagents used were as follows: anti-BNIP3L (#12396, 1:1,000 WB, 1:250 IF; Cell Signalling), anti-BNIP3 (ab109362, 1:1,000 WB; Abcam), anti-TOM20 (11802-1-AP, 1:1,000 WB; ProteinTech), anti-TOM20 (HPA011562, 1:500 IF; Sigma-Aldrich), anti-PMP70 (SAB4200181, 1:1,000 WB, 1:250 IF; Sigma-Aldrich), mouse anti‐actin (66009-1-Ig, 1:10,000; ProteinTech), anti-Cullin-2 (A302-476A, 1:2,000 WB; Bethyl Laboratories), anti-HIF1⍺ (NB100-134, 1:1,000; Novus Bio techne), anti-ATG7 (2631, 1:1,000 WB; Cell Signalling), anti-NBR1 (9891S, 1:1,000 WB; Cell Signalling), anti-LC3 (5F10, 1:200 WB; Nanotools), MLN4924 (C-1231; Chemgood), deferiprone (379409; Sigma-Aldrich), 4-PBA (21005; Sigma-Aldrich), clofibrate (C6643; Sigma-Aldrich), hydrogen peroxide (H1009; Sigma-Aldrich), oligomycin A (75351; Sigma-Aldrich), antimycin A (A8674; Sigma-Aldrich), Mitotracker Deep Red FM (M22426; Invitrogen).
6
Lipid Metabolism Regulation Assay
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Chemicals used include SB 204990 (Thermo Fisher Scientific, Paisley, UK), TOFA, C75, clofibrate, palmitic acid, and SC-236 (Sigma-Aldrich, Dorset, UK), PGD2, PGI2, and PGE2 (Cambridge Bioscience, Cambridge, UK), and SC-51322, TG-4-155, and ER-819762 (Bio-Techne Ltd., Abingdon, UK), and all were reconstituted in dimethyl sulfoxide. TXA2 (Cambridge Bioscience, Cambridge, UK) was reconstitute in ethanol. PGF2α (Cambridge Bioscience, Cambridge, UK) and etomoxir and malonyl-CoA (Sigma-Aldrich, Dorset, UK) were reconstituted in E199 medium. Anti-β-actin mouse monoclonal antibody (MAb), anti-FASN goat polyclonal antibody (pAb), anti-COX2 goat pAb, and anti-ACC rabbit MAb (Abcam, Cambridge, UK) were used for Western blot analysis. Donkey pAb to goat IgG, goat pAb to mouse IgG, and goat pAb to rabbit IgG were also purchased from Abcam, Cambridge, UK.
7
Preparation of Chemical Stock Solutions
8
Lipid Signaling Pathway Screening
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BODIPY™ 558/568 C12 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, Thermo Fischer Scientific), Hoechst (Sigma), sodium arsenite (Fischer Chemical), cycloheximide (Sigma), BODIPY™ 493/503 (ThermoFischer Scientific, D3922), Rosiglitazone (Sigma), Clofibrate (Sigma), GW501516 (Sigma), 2-bromopalmitic acid (Sigma), streptavidin-HRP (Thermo Scientific), fatty acid-free BSA (PAN Biotech), DMEM (PAN Biotech), FBS (PAN Biotech), PBS (PAN Biotech), methanol (Roth), chlorophorm (Sigma), aprotinin (Roth), leupeptin (Roth), phenylmethylsulfonyl fluoride (PMSF, Sigma), Fasnall (Sigma), and kinase screening library (10505, Cayman Chemical).
9
Antibody and Chemical Reagent Protocol
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Mouse monoclonal antibodies against FLAG (Sigma, F1804), Myc (CST, 2276S), HCV NS3 (Abcam, ab65407) and Core (Thermo, MA1-080), rabbit monoclonal antibody against HA (CST, 3724S), QPRT (Abcam, ab180930), Smurf2 (CST, 12024), rabbit polyclonal antibody against SREBP-2 (Santa Cruz, sc-13552), and fine chemicals of proteasome inhibitor MG132 (ApexBio Tech, A2585), 2,3-Pyridinedicarboxylic acid (QA, Sigma, P63204), 2,3-Pyridinedicarboxylic Acid-d3 (QA-d3, J&K Scientific Ltd, P991633), β-Nicotinamide adenine dinucleotide hydrate (NAD, Sigma, N7004), Resveratrol (Res, Sigma, V900386), Phthalic acid (PHT, Sigma, 80010), Clofibrate (Sigma, C6643) and cycloheximide (Sigma, C7698) were purchased from where indicated.
10
Stroke-Prone Rodent Model: Clofibrate Effects
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SHRSP were maintained on stroke-prone rodent diet (0.38% Na and 0.71% K, Zeigler Brothers, Gardners, PA, USA; Stier et al., 1989 (link)) and 1% NaCl drinking solution starting at approximately 7 weeks of age. Clofibrate (200 mg/kg/day, n = 7; Sigma–Aldrich, St. Louis, MO, USA) or vehicle (0.5% methylcellulose, n = 5) administered once daily by gavage, was started 3 days prior to giving SHRSP 1% NaCl drinking solution. Systolic BP (SBP) was measured weekly using tail-cuff plethysmography (CODA 2 non-invasive BP apparatus, Kent Scientific, Torrington, CT, USA). After 3 weeks of HS intake, animals were housed in metabolic cages and urine was collected for measurement of eicosanoids by Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) analysis and urinary protein excretion (UPE) by the sulfosalicylic acid turbidity method (Stier et al., 1989 (link)). Rats were then anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and kidneys were excised and sections of cortex were snap frozen in liquid N2 for Western immunoblot analysis and the remaining kidney was placed in formalin for histological evaluation.
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