Luciferase reporter assay

Cells (1×10⁵ cells/well) were seeded in 96-well plates and allowed to settle for 12 h at 37°C. Reporter plasmids (100 ng; EMD Millipore, Billerica, MA, USA) containing wild-type (CCTTTGATC; TOP flash) or mutated (CCTTTGGCC; FOP flash) T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) binding sites used to determine β-catenin transcriptional activity (18) plus 1 ng pRL-TK Renilla plasmid were co-transfected using Lipofectamine 2000 according to the manufacturer's protocol. Firefly and Renilla luciferase activity were measured 48 h following transfection using the Dual Luciferase Reporter Assay kit (Promega Corporation, Madison, WI, USA) according to manufacturer's protocol. Luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate.