Roxadustat fg 4592

Cells were treated with the following compounds dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich)⁵⁶ (link) and the final concentrations indicated: dimethyloxalylglycine (DMOG; Frontier Scientific, Logan, UT, USA; 1 mM), FG-4592 (roxadustat; Selleckchem, Houston, TX, USA; 0.1 mM) and dimethyl N-oxalyl-D-phenylalanine (DM-NOFD; Sigma-Aldrich; 1 mM). Desferrioxamine (DFX; Sigma-Aldrich; 0.1 mM) was dissolved in bidistilled water. MG132 (Sigma-Aldrich; 10 μM) was dissolved in ethanol (Sigma-Aldrich). To test the sensitivity of the oxomer against reducing agents, cell lysates were treated with dithiothreitol (DTT; Agilent Technologies) or β-mercaptoethanol (Sigma-Aldrich).
Incubations in hypoxia were performed using the InvivO₂ 400 humidified cell culture workstation (Baker Ruskinn, Bridgend, South Wales, UK) operated with 0.2% O₂ or 1% O₂, 5% CO₂ at 37 °C as previously described, or in humidified oxygen-regulated cell culture incubators (Binder, Tuttlingen, Germany) operated with 2–8% O₂ and 5% CO₂ at 37 °C.⁵⁷ (link) ”normoxia” refers to the air oxygen level in the gas phase within a humidified cell culture incubator at 500 m altitude (18.5% O₂).⁵⁸ (link)

Antibodies against AGTR1 (catalog ab124734, 1:1000), AGTR2 (catalog ab92445, 1:1000), eNOS (catalog ab199956, 1:1000), p-eNOS (catalog ab215717, 1:1000), and HIF1α (catalog ab179483, 1:1000) were bought from Abcam. Antibodies against β-actin (catalog ap0060, 1:2000) were purchased from Bioworld. Reactive oxygen assay kits were purchased from Biyuntian. TBARs assay kits were purchased from Cayman. The nitrate oxide/nitrite (NOx) assay kits were purchased from Cayman. L-NAME was purchased from MCE. FG-4592 (Roxadustat), the HIF prolyl hydroxylase inhibitor, was purchased from Selleck Chemicals.

Roxadustat (FG-4592) was purchased from Selleck (Shanghai, China) and dissolved in a mixed solvent containing 5% DMSO, 40% PEG-300, 5% Tween-80 and 50% ddH₂O according to Selleck in vivo usage instructions for mouse intraperitoneal injection. Rox powder was stored at −20 °C in silver paper to avoid light and ensure dryness, with its solvent prepared immediately prior to use. Rox was dissolved in solvent to reach a concentration of 100 mM and then diluted to concentrations of 5–100 μmol/L with cell culture medium for cell assays. METH was obtained from Shanghai Standard Biotech Co., Ltd. (Shanghai, China) and diluted with saline for i.p. injection (2.0 mg/kg). Morphine was purchased from Shenyang First Pharmaceutical Factory (Shenyang, China) and dissolved in saline for i.p. injection (5.0 or 20.0 mg/kg). Deferiprone (DFP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethanesulfonyl fluoride (PMSF) was purchased from Cell Signaling Technology (Danvers, MA, USA). Other common reagents were purchased from Sinopharm (Beijing, China).