Measuring Wnt/β-Catenin Signalling Activity

Wnt/β-catenin signalling activity was measured using a β-catenin firefly luciferase reporter plasmid containing TCF/LEF binding sites (TOP flash) (Addgene, Cambridge, MA, USA, Catalog #12456). The β-catenin reporter plasmid containing mutated TCF/LEF binding sites (FOP flash) (Addgene, Catalog #12457) served as negative control. As described previously,^{32 (link)} cells were transfected with β-catenin reporter plasmid and pRL-TK renilla luciferase reporter vector (Promega, Fitchburg, WI, USA, catalog #E2241), as an internal control for transfection efficiency, using the Lipofectamine LTX reagent (Life Technologies, catalog #15338030). Simultaneously, siRNA and pENTR4 plasmid vector were respectively transfected into CRC cells and CCD841 CoN cells. After transfection, cells were maintained in serum-free conditions for 2 to 3 days until they were examined using a Dual-luciferase Reporter Assay system kit (Promega, catalog #E1910) and a microplate reader (Corona Electric, Lethbridge, Canada, catalog #SH-9000), following the manufacturers’ instructions. Reporter activities were reflected by the luminescence intensity. Data were normalised by the value of pRL-TK renilla luciferase activity.