Mouse fibroblast cell lines, 10T1/2 (ATCC CCL-226) and NIH/3T3 (hereafter referred to as 3T3, ATCC CRL-1658) were maintained in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Marlborough, MA). In hypoxia experiments, cells were placed in an incubator with 21% O2 or in a hypoxia chamber (INVIVO2 200, Ruskinn Technology Ltd., Bridgend, UK) with 0.5% O2 for indicated duration. Cellular RNA was harvested by adding the TRIzol reagent (Invitrogen) or RNeasy mini kit (Qiagen, Valencia, CA) immediately after cells were taken out of the incubator and the supernatant was removed for storage. In roxadustat (MedChemExpress LLC, Monmouth Junction, NJ) experiments, cells were washed with 1 × PBS and renewed culture medium with or without roxadustat (50 µM). Cellular RNA was harvested at indicated time points. In TGF-β1 stimulation experiments, cells were washed with 1 × PBS and renewed culture medium with or without 5 ng/mL TGF-β1 (R&D Systems, Minneapolis, MN) in the presence of 5 µg/mL TGF-β receptor-1 (TGF-βR1) activin receptor-like kinase-5 (ALK5) inhibitor (ALK5i) SB431542 (Tocris Bioscience, Bristol, UK) or vehicle. Cellular RNA were harvested at indicated time points.
Roxadustat
Manufactured by MedChemExpress
Sourced in United States
Roxadustat is a hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor. It is a lab equipment product used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3phosphoethanolamine n folate polyethylene glycol 2000 dspe peg2000 folate, by Avanti Polar Lipids (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Rifampin, by Merck Group (1 mentions)
Quinidine, by Merck Group (1 mentions)
Molidustat, by MedChemExpress (1 mentions)
Daprodustat, by MedChemExpress (1 mentions)
Vadadustat, by MedChemExpress (1 mentions)
Ketoprofen, by Merck Group (1 mentions)
100xneaa, by Thermo Fisher Scientific (1 mentions)
4 protocols using roxadustat
1
Hypoxia and TGF-β1 Signaling in Fibroblasts
2
Synthesis and Characterization of PHD2 Inhibitors
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General chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and J.T. Baker (Radnor, PA, USA). 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG2000-folate) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). IOX2 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were from Sigma-Aldrich. Calcium (II) acetate (Ca(OAc)2) and ethylenediaminetetraacetic acid (EDTA) were from J.T. Baker (Radnor, PA, USA). Dimethyloxallyl glycine (DMOG) and vadadustat were from AbMole BioScience (Houston, TX, USA). Roxadustat was from MedChemExpress (Monmouth Junction, NJ, USA). All three PHD2 inhibitors were dissolved in dimethyl sulfoxide (DMSO) as stock solutions and diluted for use. KB and HeLa cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).
3
Hepatocyte-based Compound Solubility Evaluation
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Daprodustat, enarodustat, molidustat, roxadustat, vadadustat were purchased from MedChemExpress (Monmouth Junction, NJ). Desidustat was purchased from AmBeed (Arlington Heights, IL). Rosuvastatin was purchased from Sequoia Research Products Ltd.
(Oxford, UK). Rifampin, rifamycin SV, quinidine, ketoprofen were purchased from Sigma-Aldrich (St. Louis, MO). Cryopreserved human plateable hepatocytes (Hu8246, Caucasian, female 37 years old and HH1062, female 33 years old) were purchased from Life Techlologies (Carlebad CA) and In Vitro ADMET Laboratories (Columbia, MD). Cryopreserved cynomolgus hepatocytes (10106012) was purchased from In Vitro ADMET Laboratories (Columbia, MD).
InVitroGro-HT, CP and HI hepatocyte media were purchased from Celsis (Baltmore, MD). MDCKII-LE cells were from Pfizer (Groton, CT). MEMα, FBS, 100x NEAA, 100x GlutaMAX ® and 1% of 10,000 U/mL penicillin-10,000 µg/mL streptomycin were purchased from Gibco Life Technology (Waltham, MA). Millicell-96 insert plates and BSA were purchased from MilliporeSigma (Burlington, MA).
(Oxford, UK). Rifampin, rifamycin SV, quinidine, ketoprofen were purchased from Sigma-Aldrich (St. Louis, MO). Cryopreserved human plateable hepatocytes (Hu8246, Caucasian, female 37 years old and HH1062, female 33 years old) were purchased from Life Techlologies (Carlebad CA) and In Vitro ADMET Laboratories (Columbia, MD). Cryopreserved cynomolgus hepatocytes (10106012) was purchased from In Vitro ADMET Laboratories (Columbia, MD).
InVitroGro-HT, CP and HI hepatocyte media were purchased from Celsis (Baltmore, MD). MDCKII-LE cells were from Pfizer (Groton, CT). MEMα, FBS, 100x NEAA, 100x GlutaMAX ® and 1% of 10,000 U/mL penicillin-10,000 µg/mL streptomycin were purchased from Gibco Life Technology (Waltham, MA). Millicell-96 insert plates and BSA were purchased from MilliporeSigma (Burlington, MA).
4
Aging and Hypoxia Mimetics in Mice
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All the mice were maintained in the central animal facility (CDP) of University Medical Center Groningen (UMCG) under standard conditions. All the experiments were approved by the Central Authority for Scientific Procedures on Animals (CCD) in Netherlands. For analyses of skin and liver, samples from naturally aged C577BL/6J mice were obtained from the mouse clinic for Cancer and Aging, University Medical Center Groningen (UMCG). For all other aging experiments, p16-3MR mice were used (Demaria et al., 2014) . For experiment with hypoxia mimetics, 3,4 dihydroxybenzoic acid (Sigma-Aldrich, Cat# 37580) was dissolved in PBS and administered by daily I.P. Injection (10 mg/kg) for 7 consecutive days. Roxadustat (MedChem Express, Cat# HY-13426) was dissolved in vehicle (5% DMSO + 95% corn oil) and administered by i.p. injection (30 mg/kg) every other day for 3 times. All animals were bred in house. Aged mice were 24 months or older. Young control mice and doxo treated mice were between 3-6 months old (early adulthood). All mouse experiments were performed using mixed gender.
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