For overexpression experiments, HepaRG cells were pre-incubated for 1 h with 8 μg/ml polybrene (Sigma-Aldrich) and then transduced with concentrated CYP4A retroviruses at a multiplicity of infection of 3. For the knockdown experiment, HepaRG cells were transfected with siRNAs for CYP4A11 and CYP4A22 (Bioneer, Daejeon, Korea) using Dharmafect I transfection reagent (Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions. For the HET0016 study, cells were pre-treated with 5 μM HET0016 (Cayman, Ann Arbor, MI, USA) for 6 h before replacing the culture medium with 50 mM glucose and 125 μM palmitate, and the treatment was continued for 5 days.
Het0016
Manufactured by Cayman Chemical
Sourced in United States
HET0016 is a chemical compound used in various laboratory applications. It serves as a core functional component for research and analysis purposes. The detailed specifications and intended use of this product are not provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sq29548, by Cayman Chemical (3 mentions)
Furegrelate, by Cayman Chemical (2 mentions)
Ns 398, by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nifedipine, by Merck Group (1 mentions)
Sq 29 548, by Merck Group (1 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)
Polybrene, by Merck Group (1 mentions)
Stattic, by Merck Group (1 mentions)
Sdf 1, by R&D Systems (1 mentions)
Recombinant human macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse vegf, by R&D Systems (1 mentions)
20 hete, by Cayman Chemical (1 mentions)
Recombinant mouse il 4, by Thermo Fisher Scientific (1 mentions)
F4 80 antibody sc 377009, by Santa Cruz Biotechnology (1 mentions)
Zoledronic acid za, by Merck Group (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
A83 01, by Merck Group (1 mentions)
Tgf β, by R&D Systems (1 mentions)
13 protocols using het0016
1
Overexpression and Knockdown of CYP4A in HepaRG Cells
2
Vasomotor Responses of Middle Cerebral Artery
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Diameter responses of MCA were obtained in response to arachidonic acid (AA) (10−7, and 10−8 mol/L), and cytochrome P450 o-hydroxylases (CYP450 4A) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE, 10−7 mol/L) and 9,11-Dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619, 10−6; 10−7 mol/L), a synthetic analog of the PGH2 acting on the thromboxane-prostanoid (TP) receptors, as an agonist in various conditions.
Administration of Inhibitors: Diameter responses of MCA were obtained in the absence and presence of N-(4-butyl-2-methylphenyl)-N’-hydroxy-methanimidamide (HET0016, 10−6 mol/L an inhibitor of CYP450 4A producing 20-HETE [12 (link)] for 30 min to elucidate its role.
Additionally, we used [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ 29,548; 10−6 mol/L) aTP receptor blocker for 30 min to elucidate the role of this receptor in the vasomotor responses of MCA.
At the end of the experiments, the passive diameters were measured in the presence of Ca2+-free physiological salt solution (PSS) containing dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nifedipine 10−5 mol/L).
All drugs were purchased from Sigma Aldrich (St Louis, MO, USA), except SQ 29,548 and HET0016 (Cayman Chemical Company, Ann Arbor, MI, USA).
Administration of Inhibitors: Diameter responses of MCA were obtained in the absence and presence of N-(4-butyl-2-methylphenyl)-N’-hydroxy-methanimidamide (HET0016, 10−6 mol/L an inhibitor of CYP450 4A producing 20-HETE [12 (link)] for 30 min to elucidate its role.
Additionally, we used [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ 29,548; 10−6 mol/L) aTP receptor blocker for 30 min to elucidate the role of this receptor in the vasomotor responses of MCA.
At the end of the experiments, the passive diameters were measured in the presence of Ca2+-free physiological salt solution (PSS) containing dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nifedipine 10−5 mol/L).
All drugs were purchased from Sigma Aldrich (St Louis, MO, USA), except SQ 29,548 and HET0016 (Cayman Chemical Company, Ann Arbor, MI, USA).
3
Synthesis and Characterization of HET-TK-SA Prodrug
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TK-SA was synthesized from thioketal (TK) and stearyl alcohol (SA) by an N1-((ethylimino)methylene)-N3/4-dimethyla-minopyridine (EDC/DMAP; Aladdin Industrial, China) esterification reaction, and then HET0016 (Cayman, USA) was conjugated to it by the same reaction system; the prodrug HET-TK-SA was synthesized, the target product HET-TK-SA obtained by separation purification by silica gel column chromatography, monitored by thin layer analysis, collected by spin evaporation, and the conjugate characterized by 1H nuclear magnetic resonance (1H NMR). The synthetic route of the HET0016 prodrug is shown in Figure 2A .
4
Pharmacological Screening of Vasoactive Agents
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The drugs used were acetylcholine (ACh) chloride, noradrenaline (NA) hydrochloride, 1400W, indomethacin, NS-398 (Sigma-Aldrich, Schnelldorf, Germany), SQ-29548, furegrelate, HET0016 (Cayman Chemical, Ann Arbor, MI, USA), and tranylcypromine (USP, Twinbrook, Rockville, Italy).
According to their solubility, the chemicals were dissolved as follows: 1400W in methanol; HET0016, SQ-29548, and indomethacin in ethanol; and NS-398 in DMSO. NA was dissolved in a mixture of sodium chloride + ascorbic acid (0.9% and 0.01% w/v). Other drugs were prepared in distilled water.
The stock solutions (10 mM) were maintained at −20 °C, and appropriate dilutions were made in a Krebs–Henseleit buffer (KH buffer: mM; NaCl 115, CaCl2 2.5, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1 at pH 7.4) on the day of the experiment. The maximal solvent concentration in organ baths was less than 0.01% (v/v).
According to their solubility, the chemicals were dissolved as follows: 1400W in methanol; HET0016, SQ-29548, and indomethacin in ethanol; and NS-398 in DMSO. NA was dissolved in a mixture of sodium chloride + ascorbic acid (0.9% and 0.01% w/v). Other drugs were prepared in distilled water.
The stock solutions (10 mM) were maintained at −20 °C, and appropriate dilutions were made in a Krebs–Henseleit buffer (KH buffer: mM; NaCl 115, CaCl2 2.5, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1 at pH 7.4) on the day of the experiment. The maximal solvent concentration in organ baths was less than 0.01% (v/v).
5
Rat Model of Cerebral Endothelial Dysfunction
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Four to six-week-old male Sprague-Dawley (SD) rats (body weight, 180–260 g) (Shanghai SLAC Laboratory Animal Corporation, Shanghai, China) were studied. Rats were housed in a 12/12 h light/dark cycle environment and with provided food and water. This study was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Biomedical Engineering. Antibodies against MMP-9, occludin, ZO-1, and anti-phospho-JNK rat polyclonal antibody and anti-phospho c-Jun rat polyclonal antibody were purchased from Abcam, Inc. (Abcam, England). HET0016 was purchased from Cayman Chemical Company (Ann Arbor, MI, United States).
6
AGEs-Induced Metabolic Dysregulation in Mice
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Adult male C57BL/6 mice (6 to 8 weeks) weighing from 16 to 20 g were obtained from the Animal Center of Dalian Medical University (Dalian, China). All procedures were performed according to the Institutional Animal Care and Use Committee guidelines and approved by the Institutional Ethics Committee. The mice were kept in a specific pathogen-free grade animal facility with a 12-h light-dark cycle. Thirty-six adult male C57BL/6 mice were randomly divided into four groups (n = 9): 1) BSA control group (Con), 2) Con+HET0016 (Cayman Chemical, USA) group, 3) AGEs group, and 4) AGEs+HET0016 group. All C57BL/6 mice underwent intragastric administration of AGEs (500 mg/kg/day) or the same dosage of BSA as a control for 60 consecutive days. At 14 days before the end of the experiments, mice received intraperitoneal injections of the specific CYP4A inhibitor, HET0016 (2.5 mg/kg/d) in the Con+HET0016 and AGEs+HET0016 groups or vehicle in the BSA control and AGEs groups (Jiang et al., 2004 (link); Thomas et al., 2005 (link); Cai et al., 2014 (link); Guo et al., 2015 (link); Wang et al., 2019 (link)).
7
Macrophage-Targeted Therapy for Angiogenesis
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HET0016, DDMS and 20-HETE were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Human recombinant macrophage colony-stimulating factor (M-CSF), mouse recombinant IL-4 and IL-13 were purchased from Pepro Tech (Rocky Hill, NJ, USA). Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK). Stattic (a STAT3 inhibitor) was purchased from Merck (Darmstadt, Germany). Clodronate (Clod) was purchased from Roche Diagnostics (Indianapolis, IN, USA). Zoledronic acid (ZA) and A83-01 (a TGF-β receptor inhibitor) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Neutralizing antibodies against TGF-β (MAB7346), VEGF (AF-493) and SDF-1 (MAB310) were purchased from R&D Systems. Neutralizing antibody against PDGF-B (ab34074), mCherry (ab125096) antibody and CD68 (ab955) antibody were purchased from Abcam (Cambridge, MA, USA). Neutralizing antibody against MMP-9 (MA5-13595) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). F4/80 antibody (sc-377009) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
8
Biochemical Pathway Modulation
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ATP and H2O2 was purchased from Wako (Tokyo, Japan). LPS was purchased from Invivogen (San Diego, CA, USA). Aspirin was purchased from Tokyo Kasei (Tokyo, Japan). 1-Aminobenzotriazole, HET0016, Zileuton, PD146176, Celecoxib, MS-PPOH, ML355, and Triacsin C were purchased from Cayman (Ann Arbor, MI, USA).
9
Pharmacological Reagents for Research
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D-Methamphetamine (D-meth), L-methamphetamine (L-meth), L-cycloserine, myriocin, thalidomide, cimetidine, quinidine, SKF-525A and clotrimazole were purchased from Sigma Aldrich (St. Louis, Missouri). Fumonisin B1 (FB1), C6 and C8 ceramide, and HET-0016 were from Cayman Chemicals (Ann Arbor, Michigan). 5′-aminosalicylic acid and JSH-23 were from Santa Cruz Biotechnology (Santa Crux, CA).
10
Vascular Effect of Bioactive Compounds
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Acetylcholine (chloride), indomethacin, noradrenaline (hydrochloride), and NS-398 were obtained from Sigma-Aldrich (St. Louise, MO, USA); copper as carbonate (purity ≥ 99%) from Poch (Gliwice, Poland); SQ-29,548, furegrelate, and HET0016 from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions (10 mM) of these drugs were prepared in distilled water, except for noradrenaline, which was dissolved in NaCl (0.9%) + ascorbic acid (0.01% w/v) solution; HET0016, SQ-29,548, and indomethacin were dissolved in ethanol; 1400 W in methanol; and NS-398 in DMSO. The solvent concentration was less than 0.01% (v/v).
These solutions were stored at −20 °C, and appropriate dilutions were made in Krebs–Henseleit solution (KH in mM: NaCl 115; CaCl2 2.5; KCl 4.6; KH2PO4 1.2; MgSO4 1.2; NaHCO3 25; glucose 11.1) on the day of the experiment.
These solutions were stored at −20 °C, and appropriate dilutions were made in Krebs–Henseleit solution (KH in mM: NaCl 115; CaCl2 2.5; KCl 4.6; KH2PO4 1.2; MgSO4 1.2; NaHCO3 25; glucose 11.1) on the day of the experiment.
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