Male spontaneously hypertensive rats (SHR/NCrl) and normotensive Wistar-Kyoto rats (WKYs) in four age groups were obtain from Charles River Laboratories and housed in controlled 12 h light/dark conditions, in a constant temperature of 21 °C, with ad libitum access to water and food. Part of the animals were randomly assigned to groups treated for 30 days with GKT137831 (CAS 1218942-37-0, Adooq Bioscience LLC, USA), ML171 (CAS 6631-94-3, Merck, Germany) or placebo mixed in food (both at 60 mg/kg/day [18 (link)]). Blood pressure was measured using non-invasive tail cuff plethysmography using BP2000 Blood Pressure Analysis System (Visitech Systems Inc.), following 7 days of training period as described before [26 (link)]. At the end of experiment, rats were heparinised and euthanized using CO2 inhalation and perfused via the left ventricle of the heart using cold PBS to ensure blood cells are not contributing to organ flow cytometric analysis. All in vivo work was performed in accordance with the Animals Scientific Procedures Act 1986 and ARRIVE (Animal Research: Reporting of In Vivo Experiments) Guidelines and approved by Jagiellonian University Ethics Committee (107/2014). All experiments conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.
Ml171
Manufactured by Merck Group
Sourced in United States, Germany
ML171 is a compact, high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a durable, user-friendly interface and delivers reliable, accurate results.
Automatically generated - may contain errors
Lab products found in correlation
Vas2870, by Merck Group (3 mentions)
Oxypurinol, by Merck Group (3 mentions)
Rotenone, by Merck Group (3 mentions)
Gkt136901, by Merck Group (2 mentions)
Palmitic acid, by Merck Group (2 mentions)
Superoxide dismutase 1 sod1, by Merck Group (2 mentions)
Fatty acid free bovine serum albumin, by Merck Group (2 mentions)
Amplex ultrared, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp, by Merck Group (2 mentions)
Wistar kyoto rats, by Charles River Laboratories (1 mentions)
Shr ncrl, by Charles River Laboratories (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Uo126, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Diphenyleneiodonium chloride dpi, by Merck Group (1 mentions)
Nbp2 29328, by Novus Biologicals (1 mentions)
Pkc inhibitor go 6976, by Merck Group (1 mentions)
Nox2ds tat, by AnaSpec (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
13 protocols using ml171
1
Blood Pressure Regulation in Hypertensive Rats
2
Murine Macrophage Activation by Biglycan
3
Measuring NADPH Oxidase Activity in Retina and RPE/Choroid
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NADPH oxidase activity was measured both in retina and RPE/choroid homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [43 (link)]. To confirm the source(s) of superoxide anion (O2•−), homogenate samples were preincubated for 5 min at 37 °C with the following inhibitors at 0.1 mmol/L: diphenyleneiodonium, DPI (inhibitor of flavoproteins; Sigma-Aldrich, Madrid, Spain); oxypurinol (inhibitor of xanthine oxidase; Sigma-Aldrich, Madrid, Spain); and rotenone (mitochondrial chain inhibitor of electron transport; Sigma-Aldrich, Madrid, Spain). Following the same protocol, the inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, Madrid, Spain, 492000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, Madrid, Spain, 175226) and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, Madrid, Spain, 5340320001) were used to explore the relative contribution of each NOX isoform in O2•− production [35 (link)]. Hydrogen peroxide (H2O2) levels were measured in retina homogenates by AmplexTM Red hydrogen peroxide/peroxidase assay kit (A22188, ThermoFisher Scientific, Invitrogen, Spain) following the manufacturer’s instructions. Absorbance readings were obtained in 96-well plates at 560 nm. All measurements referred to the samples’ protein content, and results were always expressed as relative to the control group.
4
Quantifying Cellular Oxidative Stress
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Amplex UltraRed was from ThermoFisher (Cat No. A36006); horseradish peroxidase (HRP) was from Sigma (Cat No. P8125); superoxide dismutase 1 (SOD1) was from Sigma (Cat No. S7571); NOX1/4 inhibitor ML171 was from Sigma (Cat No. 492002); S1QEL2.1 and S3QEL1.2 were provided by Calico Life Sciences LLC (South San Francisco, CA) and AbbVie Inc. (Chicago, IL). Palmitic acid (PA) was from Sigma (Cat No. P5585); fatty acid-free bovine serum albumin (BSA) was from Sigma (Cat No. A7030).
5
Lutein Dosing and NADPH Oxidase Inhibition
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Lutein, purchased from Cayman Chemical Co. (Catalog No. 10010811, Ann Arbor, MI, USA), was dissolved in dimethyl sulfoxide (DMSO) according to the product information (Cayman Chemical Co, Ann Arbor, MI, USA), aliquoted, and stored at −80 °C. Final concentrations of 5, 10, or 20 μM were applied to the cells. NADPH oxidase 1 inhibitor ML171 and etoposide were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO. Antioxidant NAC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water. For each experiment, cells incubated with DMSO alone (less than 0.3%) served as the control.
6
Corneal NADPH Oxidase Activity Assay
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NADPH oxidase activity was measured in cornea homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [48 (link)]. Potential sources of superoxide anion (O2.−) production in corneal samples were discriminated at 37 °C after a 5-min preincubation with 0.1 mmol/L DPI, oxypurinol, or rotenone (respective inhibitors of flavoproteins, xanthine oxidase, and mitochondrial electron transport chain; Sigma-Aldrich, Madrid, Spain). Similar protocols were followed when using an inhibitor of NOX1/4 (0.1 µmol/L GKT136901; Sigma-Aldrich, 492,000), specific NOX1 inhibitor (0.5 µmol/L ML171; Sigma-Aldrich, 175,226), and the pan-NADPH oxidase inhibitor (10 µmol/L VAS2870; Sigma-Aldrich, 5,340,320,001) to determine the relative contribution of each NOX isoform in total O2.− production. All measurements were referred to the samples’ protein content, and results were always expressed as relative to those in the control group.
7
Measurement of Nox-Derived Reactive Oxygen Species
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RPMI 1640 with Glutamax, DMEM/F12 (1:1), Hanks' buffered salt solution (HBSS), fetal bovine serum (FBS), and Amplex red were purchased from Invitrogen, Paisley, UK. Pest (penicillin, streptomycin), neomycin, ionomycin, phorbolmyristateacetate (PMA), diphenyleneiodoniumchloride (DPI), dapsone, ML-171, Phox-I2, xanthine, hypoxanthine, xanthine oxidase, DMSO, DPPH (2,2-diphenyl-1-1picrylhydrazyl), Tween20, Sucrose, flavin adenine dinucleotide (FAD), Phosphatidic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), horseradish peroxidase (HRP) and NADPH were purchased from Sigma–Aldrich. HEK293 overexpressing Nox4 (CJ Nox4) cells were purchased from Redoxis, Lund, Sweden. HEK 293 cells expressing Nox5 and CHO cells expressing Nox1 were a kind gift from the Vincent Jaque Center Medical Universitaire, Geneva, Switzerland. GKT136901, a Nox1/Nox4 selective inhibitor, was a kind gift from prof. Harald HH Schmidt (Maastricht University, Netherlands).
8
Analyzing Platelet Signaling Cascades
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Anti-FAK, anti-Pyk2, the anti-phosphotyrosine antibody, 4G10, and HRP-conjugated goat anti-mouse and mouse anti-rabbit light chain specific IgGs were all obtained from Millipore (Lake Placid, NJ, USA); normal rabbit and mouse IgGs and RGD peptide were from Santa Cruz (CA, USA), while anti-PLCγ2 and anti-Hic-5 were from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-Rac1 was from Tebu-Bio (Peterborough, UK). Cross-linked collagen related peptide (CRP) was purchased from Prof. Richard Farndale (Dept of Biochemistry, Cambridge University, UK). The pharmacological inhibitors, PF-573228 (hereafter referred to as PF-228), PP2, Wortmannin, EHT-1864, U73122, GF109302× and the Ca2+ chelator, BAPTA, were from Tocris Bioscience (R&D Systems Europe, UK). Tyrphostin A9 was from Calbiochem. ML171 (2-acetylphenothiazine), and BAY61-3606 (hereafter referred to as BAY) were purchased from Sigma Aldrich (St. Louis, MO, USA).
9
Measurement of Placental and Renal NADPH Oxidase
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Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and superoxide anion (O2●−) sources were measured in placenta and renal cortex homogenates by lucigenin-enhanced chemiluminescence [21 (link)]. Samples were preincubated for 5 min at 37 °C with the following inhibitors of potential O2●−sources (0.1 mmol/L; Sigma-Aldrich): DPI (inhibitor of flavoproteins); oxypurinol (inhibitor of xanthine oxidase); and rotenone (inhibitor of the mitochondrial electron transport chain). Different inhibitors of NADPH oxidase (Sigma-Aldrich) were also used as follows to explore the relative contributions of each NOX isoform in O2●− production: specific NOX1 inhibitor (0.5 µmol/L, ML171, cat. # 175226); NOX1/4 inhibitor (0.1 µmol/L, GKT136901, cat. # 492000); and a pan-NADPH oxidase inhibitor (10 µmol/L, VAS2870, cat. # 5340320001). All measurements were normalized to the protein content in the samples, and results were expressed in relation to the NP group.
10
Nox1 Inhibitor Signaling Pathway
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The Nox1 inhibitor, ML171 (2-acetylphenothiazine), was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in DMSO. Apocynin, aspirin and phorbol 12-myristate 13-acetate (PMA) were purchased from Tocris (R&D systems, Inc.) and reconstituted in DMSO. The final concentration of the vehicle DMSO in all experiments was 0.1% (except for studies with aspirin which contained 0.5% DMSO). Anti-ERK 1/2, anti-pERK 1/2Thr 202/Tyr 204, anti-Akt, anti-pAktSer 473, anti-p38 and anti-P-p38Thr 180/Tyr 182 antibodies were from Cell Signaling Technology (Boston, MA, USA). HRP-conjugated anti-rabbit light chain-specific IgG was from Millipore (Lake Placid, NJ, USA). Crosslinked collagen related peptide (CRP) was purchased from the Department of Biochemistry, University of Cambridge, UK. Fibrillar Horm collagen was from Nycomed (Munich, Germany).
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