HEK-293 (CRL-1573; ATCC, Manassas, VA, USA) and SW-1353 (HTB-94; ATCC) cells were cultured in monolayer in 2 ml DMEM supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA). Cells (0.3 million) were plated in each well of six-well plates and transfected 4–6 h later with a mixture made of 100 μl DMEM, 3 μl FuGENE6 (Promega) and 1 μg plasmids. The latter included 500 ng of reporter plasmid (Col2a1 [5x48]-p89Luc (36 (link)), Acan [4xA1]-p89Luc (37 (link)), pG5Luc (Promega), 6FXO-p89Luc (38 (link)) or TOP-Flash (39 (link))), 100 ng of pSVβGal plasmid (reporter used to measure transfection efficiency) (40 (link)), and 400 ng of expression plasmids (various combinations of empty pCDNA 3.1, pCDNA 3.1-SOXE, pCDNA 3.1-SOX17, pCDNA 3.1-SOX5 and pCDNA 3.1-SOX6, pBind-GAL4DBD/SOXE, or constitutively stabilized β-catenin/CS2 plasmid (37 (link),41 (link))). Cell extracts were prepared in Tropix lysis buffer (0.2% Triton X-100, 100 mM potassium phosphate, pH 7.8, 1 mM DTT) 20–24 h after the start of transfection and assayed for luciferase and β-galactosidase activities using the Dual-Light Luciferase & β-Galactosidase Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA) and a GloMax Explorer Multimode Microplate Reader (Promega). Reporter activities were normalized for transfection efficiency by calculating the ratios of luciferase versus β-galactosidase activities.