Recombinant human ifnγ

Human primary vascular ECs were isolated as we previously reported [9 (link),20 (link)] and used between passages 2 and 5. ECs were cultured in an endothelial cell basal medium (ECBM) supplemented with 5% fetal calf serum (FCS), 0.004 mL/mL ECGS/heparin, 0.1 ng/mL hEGF, 1 ng/mL hbFGF, 1 µg/mL hydrocortisone, 50 µg/mL gentamicin and 50 ng/mL amphotericin B (C-22010, PromoCell, Heidelberg, Germany). ECs isolated from different donors (n = 5) were used in replicate experiments to ensure HLA allele diversity and avoid HLA-type-dependent effect. For activation, confluent EC monolayers were starved overnight and incubated with recombinant human IFNγ (100 U/mL, BioTechne, Abingdon, UK) for the indicated period of time in ECBM supplemented with 2% FCS. When applicable, cells were preincubated with natural or commercial compounds (1–20 µM) or diluent alone (DMSO 1/1000) for 1 h before further incubation with TNFα (100 U/mL) or IFNγ (100 U/mL) for 18 h or 48 h. Negative controls were assessed using diluent alone (DMSO 1/1000) but also structurally irrelevant biomolecules such as zoledronic acid and simvastatin.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Millipore Sigma (Billerica, MA, USA). Recombinant human TNF-α and recombinant human IFN-γ were purchased from Bio-Techne Ltd. (Abingdon, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Primary antibodies against p-IKK α/β (cat no. 2697), NF-κB p65 (cat no. 8242), p-Akt (cat no. 9271), and ICAM-1 (cat no. 4915) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against IKK α/β (cat no. 7607), p-IκB-α (cat no. 8404), IκB-α (cat no. 203), Akt1/2/3 (cat no. sc-8312), PARP (cat no. sc-9542), α-tubulin (cat no. sc-8035), Filaggrin (cat no. sc-66192), VCAM-1 (cat no. sc-1504), E-selectin (cat no. sc-5262), and β-actin (cat no. sc-81178) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories, Inc. (West Grove, PA, USA). The histamine ELISA kit was obtained from Enzo life Sciences, Inc. (Farmingdale, NY, USA). The ELISA kits for TNF-α and IL-6 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).