Vasculife engs

Human gastric carcinoma cell lines (NCI-N87, YCC-19, YCC-38, KATOIII, Hs746T, and MKN74), normal gastric epithelial cell line (HFE145), and normal primary human umbilical vein endothelial cell line (HUVEC) were used in this study. NCI-N87 and HUVEC cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, United States). KATOIII, Hs746T, and MKN74 cell lines were purchased from Korean Cell Line Bank (KCLB; Seoul, Korea). YCC-19 and YCC-38 cell lines were established and provided by Sun Young Rha (Yensei University, Seoul, Korea) (Kim et al., 2018 (link)). HFE145 cell line was provided by Hassan Ashktorab (Howard University, MD, United States) (Marlink et al., 2003 (link)). All cell lines were cultured with RPMI 1640 Medium (Hyclone, Logan, UT, United States) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, United States), 100 units/ml penicillin, and 100 μg/ml streptomycin (Hyclone, Logan, UT, United States). HUVECs were maintained in VascuLife EnGS (containing endothelial cell growth supplement; Lifeline Cell Technology, Frederick, MD, United States). All cultured cells were incubated at 37°C in a cell culture incubator with 5% CO₂.

To resemble the tissue basis for CAD, primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) were the preferred cell types. However, our preliminary study showed that HCAEC lacks sufficient proliferative capacity to support scalable yield of proteomic samples, particularly with regard to epitope-tagged index protein production by expression vector transfection. As an alternative, we identified a more proliferative EC type with a transcriptional profile that resembles HCAEC, human aortic endothelial cell (HAEC)^{85 (link)}. HAEC and HCASMC were then used to carry out all proteomic experiments.
HAEC and HCASMC from multiple healthy donors were pooled and maintained in VascuLife EnGS and SMC media, respectively (cell and medium purchased from Lifeline Cell Technology), and used at passage <8 for all experiments. HEK293 cell was from ATCC and maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplement and 10% fetal bovine serum (FBS; Thermo Fisher Scientific). All cell culture was maintained free of antibiotics in a humidified incubator at 37 °C with 5% CO₂.

Cos-7 and HEK293T cells (originally from ATCC) were cultured in Dubecco's modified Eagle's media (DMEM) from Sigma with 10% fetal bovine serum. Primary HUVECs (Lifeline Cell Technology, lot #1023) were cultured in human endothelial cell culture media, VascuLife EnGS (Lifeline Cell Technology, #LL-0002) (basal EnGS media, 0.2% EnGS, 5 ng ml⁻¹ rh EGF, 50 μg ml⁻¹ ascorbic acid, 10 mM L-glutamine, 1.0 μg ml⁻¹ hydrocortisone hemisuccinate, 0.75 U ml⁻¹ heparin sulfate and 2% FBS).