Caco2 Cell-Based Wnt and T3/TRα1 Signaling Assay

Caco2 cells were seeded onto 24-well plates (1 × 10⁶ cells/well) in DMEM supplemented with 10% FBS, which contains physiological concentrations of thyroid hormones [61 (link), 62 (link)]. For T3 treatments, the cells were maintained in 10% thyroid hormone-depleted FBS [61 (link)]. T3 (10^–6 M) or vehicle alone was added to the culture medium 24 hours before the end of the culture. We monitored the Wnt activity using the following vectors: Topflash or Fopflash (Upstate; 200 ng/well); pClneo-β-cateninXL (100 ng/well); EVR2-TCF4E (100 ng/well); pRL-CMV (1 ng/well; Promega). We monitored the T3/TRα1-dependent activity using the following vectors: pGl2-DR4-Luc (200 ng/well); pGS5-TRα1 (100 ng/well) (15); pRL-CMV (1 ng/well; Promega). The plasmids were transfected using the Exgen 500 transfection reagent (Euromedex). Luciferase activity was measured 48 hours post-transfection using the Dual-Luciferase Reporter Assay System (Promega). Data-graphs represent the normalization of beetle-luciferase (Wnt or TR responsive)/renilla-luciferase (not responsive) activities measured in each well, to correct for eventual differences in transfection efficiency from well to well.