TOP Flash/FOP-Flash reporter assay

The β-catenin reporter plasmid (TOP-flash) and its mutant control (FOP-flash) were constructed by Millipore Corporation (Massachusetts, USA). Cells were serum-starved overnight and co-transfected with 0.2 μg TOP flash or FOP flash expression plasmids and 0.1 μg pRL-TK (Renilla TK-luciferase vector; Promega, Madison, USA) using Lipofectamine 2000. The activities of both firefly and Renilla luciferase reporters were determined at 48 hours after transfection using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The TOP-Flash reporter activity is presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity, and the TOP/FOP ratio was used as a measure of β-catenin-driven transcription.