Hy926 cells

HUVECs (Lonza, Walkersville, MD) were culture in EGM-2 medium (Lonza). Human endothelial EA.hy926 cells (ATCC, Manassas, VA, USA) were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Biowest, Nuaillé, France).

HUVEC-derived endothelial EA. hy926 cells (ATCC, #CRL-2922) were grown at 37°C in 8% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, #10–013-CV and #10437–028) supplemented with 10% fetal bovine serum (FBS) and 20% preconditioned media as we previously described (Molinar-Inglis et al., 2021 (link)). Endothelial cell confluent monolayers were washed with phosphate buffered saline (PBS) and grown in low-serum 0.4% FBS DMEM overnight. Cells were then washed and serum starved in DMEM containing 10 mM HEPES, 1 mM CaCl₂, and 1 mg/mL bovine serum albumin (BSA) for 1 h prior to agonist, antagonist and/or inhibitor treatments at 37°C.
Endothelial EA. hy926 cells were pretreated with or without 20 nM APC for 3 h at 37°C (Prolytix, #HCAPC-0080). Cells were then stimulated with 10 ng/mL TNF-α (PeproTech, #300–01A) for the indicated times at 37°C and responses measured. To block PAR1 signaling, serum-starved cells were pretreated at 37°C with 10 µM Vorapaxar (Axon Medchem, #1755) for 1 h before APC treatment. S1PR1 signaling was inhibited by incubation with 10 µM W146 (Tocris, #3602) for 30 min at 37°C. GRK2 was inhibited by preincubating endothelial cells with 50 µM Compound (CPMD)101 (Sigma-Aldrich, #SML2784) at 37°C for 1 h in serum-free media before APC stimulation.

THP-1 cells (ATCC, Manassas, Virginia, USA) were grown in RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and supplemented with 10% FBS (STEMCELL, Cambridge, UK), Penicillin–Streptomycin (STEMCELL, Cambridge, UK), and 3.6 μL 2-Mercaptoethanol (Merck, Darmstadt, Germany) in a 5% CO₂ humidified atmosphere at 37 °C.
EA.hy926 cells (ATCC, Manassas, Virginia, USA) were grown in DMEM (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) under the same conditions, except for supplementation with 2-Mercaptoethanol.