Primary human pulmonary arterial endothelial cells (HPAECs) were purchased and propagated in EGM-2 cell culture media (Lonza). Experiments were performed at passages 4–9. HPAECs were exposed for 24 or 48 hours either to standard non-hypoxic cell-culture conditions (21% O2, 5% CO2, with N2 balance at 37°C) or to hypoxia (4% O2, 5% CO2, with N2 balance at 37°C), in a modular hypoxia chamber.
miR-199b-5p mimic (to mimic the mature form of miR-199b-5p), miR-199b-5p inhibitor (hairpin loop oligonucleotides that carry specific antisense sequences) and negative control molecules were obtained from Dharmacon (USA) and transfected into HPAECs, using Geneporter B reagent (GeneTherapy Systems), at a final concentration of 60 nM for the mimic and 100 nM for the inhibitor, according to the manufacturer’s protocol. Transfected cells were allowed to recover in normal growth media for 24 hours prior to hypoxia treatment (24 hours).
miR-199b-5p mimic (to mimic the mature form of miR-199b-5p), miR-199b-5p inhibitor (hairpin loop oligonucleotides that carry specific antisense sequences) and negative control molecules were obtained from Dharmacon (USA) and transfected into HPAECs, using Geneporter B reagent (GeneTherapy Systems), at a final concentration of 60 nM for the mimic and 100 nM for the inhibitor, according to the manufacturer’s protocol. Transfected cells were allowed to recover in normal growth media for 24 hours prior to hypoxia treatment (24 hours).