Huvec tert 2

Human White colon adenocarcinoma (Caco-2) cells (European Collection of Cell Cultures (ECACC, UK) were isolated from a primary colonic tumor in a 72-year-old White male using the explant culture technique. During routine subculture, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with the supplementation of 10% fetal bovine serum (FBS), 1% non-essential amino acid and penicillin–streptomycin solution, at 37 °C, in an incubator containing 5% CO₂. The passage number of Caco-2 cells was 25–40 in the study.
HUVEC/TERT 2 was obtained from ATCC (ATCC, Manassas, VA, USA). The cell line was isolated from the vascular endothelium of a White female patient. Cells were maintained in M199 with the supplementation of 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% amphotericin B, 2 mM glutamine (Biosera, Nuaille, France), and Endothelial Cell Growth Medium-2 (Lonza, Basel, Switzerland), at 37 °C, in a Galaxy 170R incubator under 5% CO2 (Eppendorf, Hamburg, Germany). Adhesion of the cells was support with a 0.1% gelatin solution.

Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA). Primary human umbilical vein endothelial cell (HUVEC) was purchased from Thermo Fisher Scientific (C0035C, Waltham, MA, USA) and grown in M200 medium (Thermo Fisher Scientific, M200500, Waltham, MA, USA) containing large vessel endothelial supplement (LVES) (Thermo Fisher Scientific, A1460801, Waltham, MA, USA). For transduction, Lenti-X HEK 293T cells (Clontech, 632180, CA, USA) were cultured using DMEM high glucose medium (Gibco, USA) which was supplemented with 10% FBS and 1X Antibiotic-Antimycotic. All the cells were maintained in a humidified incubation chamber with 5% CO₂ at 37 °C. Cells were treated with freshly prepared and filter sterilized Hcy (Sigma-Aldrich, H4628, USA), Cys (Sigma-Aldrich, C9768, St. Louis, MO, USA), 4-PBA (Sigma-Aldrich, P21005, USA) and TUDCA (Selleck Chemicals, S3654, Houston, TX, USA) at indicated doses. For rescue experiments, 4-PBA and TUDCA pretreatments were performed for 2 h and 14 h, respectively.

HUVEC/TERT 2 was obtained from ATCC (ATCC, Manassas, VA, USA). The cell line was isolated from the vascular endothelium of a white female patient. To culture the cells, M199 medium (Biosera, Nuaille, France) was used, which was supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% amphotericin B, 2 mM glutamine (Biosera, Nuaille, France) and endothelial cell growth medium-2 (Lonza, Basel, Switzerland). The cells were maintained in an Esco CelCulture^® CO₂ Incubator (Esco Lifesciences Group, Singapore) at 37 °C, under 5% CO₂. Before seeding, to support the adhesion of the cells, 0.1% gelatin solution was used. To create the inflammatory model, LPS (eBioscienc, San Diego, CA, USA) was added to the M199 medium to a final concentration of 100 ng/mL. The cells were divided into four groups, 24 h of incubation with basic medium (control), 24 h incubation with 100 ng/mL of LPS (LPS), 24 h incubation with 100 μg/mL of HAE (100 μg/mL HAE) and 24 h incubation with 100 ng/mL of LPS plus 100 μg/mL of HAE (LPS + 100 μg/mL HAE).