Bml ei347

Human brain pericytes (P10363), human astrocytes (P10251), human cortical
neurons (P10152) and human brain microvascular endothelial cells
(P10361) were obtained from Innoprot (Bizkaia, Spain) and maintained
in complete pericyte medium (P60121), complete astrocyte medium
(P60101), complete neuronal medium (P60157) and endothelial cell
medium (P60104), all from Innoprot (Bizkaia, Spain). Human brain
pericytes and human brain microvascular endothelial cells were seeded
in plates covered with 0.1% Gelatin Type B from Bovine Skin (G9382,
Sigma-Aldrich, San Luis, MO, USA). Human astrocytes and human cortical
neurons were seeded in Poly-L-lysine hydrobromide covered plates
(P6282, Sigma-Aldrich, San Luis, MO, USA). Cells were maintained at
37°C in an atmosphere with 5% CO₂ and 95% air. For hypoxic
conditions cells were maintained in 1% O₂ for 16 hours.
DMOG (dimethyloxaloylglycine) (BML-EI347, Enzo Life Sciences, Inc.
Farmingdale, NY, USA), a pan prolyl-4-hydroxylase inhibitor, was used
at 0.1 mM for 16 hours.

The 786-O-LUC/HIF1α/HIF1α-bHLH^∗,¹⁸ (link) G55, MDA-MB-231 and A549 cell lines, and the murine embryonic fibroblasts (MEFs) were all maintained in pyruvate-free Dulbecco’s high glucose modified Eagle’s Medium (DMEM: SH30022.01, Cytiva HyClone) supplemented with 100 units/mL of penicillin and 100 μg/mL streptomycin (P/S: DE17-602E, Lonza), 20 mM HEPES buffer (17-737E, Lonza) and 10% fetal bovine serum (FBS: SV30160.03, Cytiva HyClone). In the experiments, 786-O-LUC/HIF1α/HIF1α-bHLH^∗ cells were treated with doxycycline (1 μg/mL: D9891, Sigma-Aldrich) to induce vector expression. Cells were kept at 37°C in an atmosphere with 5% of CO₂ and 95% air for normoxic conditions. To induce hypoxia the cells were maintained at 0.5% O₂ in an Invivo2 400 workstation (Ruskinn) or exposed to dimethyloxalylglycine (DMOG, 0.1–1 mM: BML-EI347, Enzo Life Sciences, Inc.), a pharmacological inhibitor of prolyl-4-hydroxylase. All experiments were carried out in 5 mM glucose unless specifically stated.