Cell Culture and Transfection Protocol

COS-7 [34] (link)–[36] (link) and HEK cells [35] (link), [37] (link) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing L-glutamine, penicillin, streptomycin, and 10% (v/v) fetal bovine serum. Cells were transfected using Lipofectamine 2000 reagent (Invitrogen) as described previously[34] (link)–[36] (link), [38] (link), [39] (link). The HA-Nox5 HEK293 cell line was generated by using Flp Recombinase-Mediated Integration (Invitrogen)[36] (link), [37] (link). Human lung microvascular endothelial cells (HLMVEC) were purchased from Lonza, and were grown in Endothelial Growth Medium-2-Microvessel (EGM-2MV) consisting of defined growth factors and supplemented with additional FBS up to 5% final concentration (Lonza). Cells were grown at 37°C in 5% CO₂ incubator and used from passage 2–6.
Ro 32-0432 (Bisindolylmaleimide XI hydrochloride) and Gö 6976 were obtained from Sigma-Aldrich (St.Louis, MO). L-glucose and D-glucose were purchased from Thermo Fisher Scientific (Waltham, MA).