M199 medium

A549 cells (ATCC CRM-CCL-185) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich). HPMEC-ST1.6R [31 (link)] were cultured in M199 Medium (Biochrom, Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Biochrom), 5000 U/mL heparin (Biochrom), 5 μg/mL endothelial cell growth supplement (Omnilab, Bremen, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in their respective growth media at 37°C in a humidified atmosphere with 5% CO₂.
For stimulation assays, 3.5×10⁵ A549 cells and 2.5×10⁵ HPMEC per well were seeded in uncoated six well plates (Greiner, Frickenhausen, Germany). 24 h later, cells were stimulated as indicated in 1 mL fresh growth medium without antibiotics.

HUVEC were acquired from PromoCell as cryo-conserved pools (C-12203) and cultured on Corning CellBind dishes at 37 °C and 5% CO₂ in 1:1 mixed medium comprising M199 medium (BioChrom) supplemented with 10% FCS, 20 µg/ml gentamycin, 15 µg/ml amphotericin B, and ECGMII (PromoCell) supplemented with 20 µg/ml gentamycin, 15 µg/ml amphotericin B. Experiments were conducted with HUVEC passage 3–5.
HUVEC were transfected using the Amaxa nucleofection system (Lonza) according to the manufacturer’s specifications. Program U-001 was employed with the following modifications: Per cuvette 20 cm² confluent cells and 1–6 µg plasmid DNA or 400–500 pmol siRNA were re-suspended in self-made transfection buffer (4 mM KCl, 10 mM MgCl₂, 10 mM sodium succinate, 100 mM NaH₂PO₄, pH 7.4 adjusted with NaOH or HCl).