NOX1 Knockdown Modulates IL-4/IL-13 Response in HT-29 Cells

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.