Proinflammatory Stimulation of Urothelial Cells

Human non-invasive cancer urothelial cells, RT4, isolated from transitional cell papilloma (HTB-2, ATCC, Manassas, VA) were grown in 75 cm² cell culture flasks in basal media consisting of equal parts of advanced Dulbecco’s modified Eagle’s medium (A-DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (HAM) (Sigma Aldrich, St. Louis, MO, USA), supplemented with 5% fetal bovine serum (FBS), and 4 mM GlutaMAX (both Gibco, Thermo Fisher Scientific, USA). Cells repeatedly tested negative for mycoplasma infection using MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). For the experiments, RT4 cells were seeded in 6-well plates with (for IF) or without (for RNA and protein extraction and measurement of protein levels in supernatants) coverslips or 96-well plates (for viability assay) at a seeding density of 5×10⁴ cells/cm² and grown until reaching 80-90% confluency (approximately 3-4 days). To mimic a proinflammatory environment, cells were treated with 20 ng/ml human recombinant TNFα (Cayman Chemicals, Ann Arbor, MI, USA) for 24 h in serum-free basal media, as previously described (18 (link)–21 (link)). Untreated cells grown in serum-free basal media served as controls.