M50top flash

WNT activation was assessed by TOPFlash assay^{66 (link)}, after optimizing the experimental conditions for NCI-H1694 cells. WNT-responsive firefly luciferase reporter plasmid with 7 TCF/LEF binding sites (M50 Topflash; plasmid #12456), renilla luciferase (pMULE-Renilla-CMV; plasmid #62186), and mutant TOP-Flash (M51-FOPFlash; Plasmid #12457) plasmids were obtained from Addgene (Cambridge, MA, USA). Briefly, 1 × 10⁶ cells lentivirally transduced with shAPC and shScr were plated in six-well plates with media containing 10% FBS, following puromycin selection. Cells were then co-transduced with TOPFlash DNA and Renilla luciferase (10:1 ratio) using Lipofectamine 2000 from ThermoFisher (Waltham, MA, USA). Co-transduction of FOPFlash (which contains mutant TCF binding sites) and Renilla luciferase in a similar 10:1 ratio was performed as a control for this experiment. Media was exchanged after 24 h. Luciferase assay readings were obtained using the Dual Glo Luciferase assay kit from Promega on a TD-20/20 Luminometer manufactured by Turner Designs (Sunnyvale, CA, USA), according to manufacturer’s instructions at 48 h post transfection. Ratio of FOPFlash/Renilla luciferase values was subtracted from the ratio of TOPFlash/Renilla luciferase values.

Briefly, 50 pg pTK-Renilla and 100 pg M50 TOPFlash (Addgene, 12456) or M51 FOPFlash (Addgene, 12457) (Veeman et al., 2003 (link)) were co-injected with 200 pg of either fezf2 or control lacZ mRNA. Injected Xenopus embryos were collected at stage 10.5 and analysed with the DLR system (Promega). For further details see supplementary Materials and Methods.