Ht 29 human colon

HUVEC cell lines were obtained from Cell Applications Inc. (San Diego, CA, USA). They were cultured in Endothelial Cell Growth Medium (ECGM-2) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin and placed into an incubator with 5% CO₂ at 37 °C until 80% confluency was reached. Cells in the logarithmic phase were used in the assays. HT-29 human colon adenocarcinoma cell line (ATCC HTB-38), T-84 human colon carcinoma cell line (ATCC CCL-248) and SW-837 human rectum adenocarcinoma (ATCC CCL-235) were obtained from the Cell Cultures Unit of the University of Granada (Granada, Spain). Peripheral blood mononuclear cells (PBMCs) were provided by the biobank of Sistema Sanitario Público de Andalucía (SPPA) from the blood samples of healthy volunteers. Tumoral cells were cultured in darkness at 37 °C and humidified atmosphere of 5% CO₂, with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 μL/mL penicillin-streptomycin 100× and 2 mM L-glutamine. PBMCs were cultured with RPMI-1460 medium supplemented with 10% FBS.

HT-29 human colon cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in McCoy's 5A medium (Lonza, Walkersville, MD) with 10% FBS (Gemini Bio-products, West Sacramento, CA). A stable clone of HT-29 cells that expresses a scrambled NOX1 shRNA (SC cells), and two independent, clonal HT-29 cell lines that express a NOX1 shRNA producing approximately 65-70% (Si6/G6 cells) or > 90% (6A cells) reduction in NOX1 expression have been described previously [16 (link)]. WiDr, SW403, NCI-H508, and DLD-1 human colon cancer cell lines were also obtained from ATCC and were propagated in RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT) with 10% FBS. Tumor cells were cultured in a humidified incubator at 37°C in an atmosphere of 5% CO₂ in air. Parental HT-29 tumor cells, and the SC, Si6/G6, and 6A clonal variants were seeded into 60 mm tissue culture plates (Sarstedt, Inc., Newton, NC) at a concentration of 1×10⁵ cells/plate in McCoy's 5A medium containing 10% FBS. After one day in culture, cells adherent to the plates were treated with 50 ng/ml of IL-4 or IL-13; cell proliferation was determined by counting each day using a Cellometer Auto T4 Cell Counter (Nexcelom Bioscience, Lawrence, MA). Every sample was measured in triplicate; the data represent a minimum of three independent experiments.