Canonical WNT-pathway activity in the in vitro material was assessed using bioluminescence-based quantification with luciferase reporter construct (firefly luciferase cassette under the control of seven TCF binding sites, 7-TFP), as previously described (57 (link)). This reporter, which measures occupied CTNNB1 TCF/LEF-binding sites, was stably integrated into cells. Infectious lentiviral particles carrying the reporter were generated using the third generation lentiviral packaging system, as described (58 (link), 59 ); stable integration was selected using puromycin (Sigma- Aldrich) at a concentration of 2 μg/ml. Cells overexpressing WNT due to the introduction of point-mutated CTNNB1/β-catenin served as positive controls and were generated in our lab, as previously described (34 (link)).
For each measurement, cells were harvested, washed in 1x phosphate-buffered saline (PBS) and lysed according to manufacturer’s descriptions using the Dual-Light luciferase & β-galactosidase reporter gene assay system (#T1003, Life Technologies). Luminescence readout was performed at 490 nm emission wave-length on an Infinite M1000Pro plate reader (Tecan, Morrisville, NC) and normalized to β-galactosidase activity.
For each measurement, cells were harvested, washed in 1x phosphate-buffered saline (PBS) and lysed according to manufacturer’s descriptions using the Dual-Light luciferase & β-galactosidase reporter gene assay system (#T1003, Life Technologies). Luminescence readout was performed at 490 nm emission wave-length on an Infinite M1000Pro plate reader (Tecan, Morrisville, NC) and normalized to β-galactosidase activity.
