Humidified oxygen regulated cell culture incubators

Cycloheximide (CHX; Sigma-Aldrich) was dissolved in ethanol; desferrioxamine (DFX; Sigma-Aldrich), (+)-sodium l-ascorbate (Sigma-Aldrich), 2-oxoglutarate (Sigma-Aldrich), diethyl 2-oxoglutarate (DE-2OG; Sigma-Aldrich) and iron (II) sulfate (Sigma-Aldrich) in H₂O; and dimethyloxalylglycine (DMOG; Frontier Scientific, Logan, UT, USA) and FG-4592 (Roxadustat; Selleckchem, Houston, TX, USA) in dimethylsulfoxide (DMSO, Sigma-Aldrich). Hypoxic incubations were performed using the InvivO₂ 400 humidified cell culture workstation (Baker Ruskinn, Bridgend, South Wales, UK) operated at 0.2% O₂ and 5% CO₂ as described previously [29 (link)] or in humidified oxygen-regulated cell culture incubators (Binder GmbH, Tuttlingen, Germany) operated at 1%–8% O₂ and 5% CO₂. If not otherwise indicated, “normoxia” refers to the standard oxygen concentration in the gas phase within a cell culture incubator at 500 m altitude (18.5% O₂) and “hypoxia” to 0.2% O₂ [30 (link)].

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)