TOPflash luciferase reporter assay

Cells from ovarian cancer cell lines were seeded 40,000 per 50 μL in 96 well plates. Following 24 h, cells were transfected with 200 ng of TCF/LEF luciferase reporter (TOPFlash) (plasmid courtesy of Dr. Randall Moon, Upstate Biotechnology, Lake Placid, NY). Cells were transfected using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM (Gibco/Invitrogen) per the manufacturer's instructions. After 6 h, cells were treated with 1 μM niclosamide, analog 11, or analog 32 and assayed for luciferase activity with or without Wnt3a ligand 24 h post-treatment. Luciferase activity was measured using a Turner 20/20 luminometer (Promega, Madison, WI) and was normalized to the total protein concentration as reported previously [23]. The luciferase activity was normalized to untreated control and represented as the mean ± SE for a minimum of 3 replicates. Recombinant Human Wnt-3a Protein (100 ng/ml) R&D Systems Inc. Minneapolis, MN (Catalog #5036-WN-010) was used to stimulate β-catenin driven Wnt signaling.