TOP/FOP flash assay

TOP/FOP flash assay is a luciferase reporter assay which is used to assess the activity of β-catenin and T-cell factor (TCF) signaling. The cells (3×104 cells/well in 24-well plates) were transfected with 0.1 g of TOPflash or FOPflash (Upstate Biotechnology, Lake Placid, NY, USA) and 5 ng of pRL-SV40 (Promega, Madison, WI, USA) using Lipofectamine 2000 reagent (Invitrogen). After 24 h, the cells were switched to normal complete medium with or without 10 µm nicotine for another 24 h. Then, the cells were lysed, and the luciferase activity was determined according to the manufacturer's recommendations. The luciferase activity of each sample was normalized with its respective Renilla luciferase activity.