TOP-flash assay

The TOP-flash Assay was conducted as previously described [35] with minor modifications. Briefly, 1.5 × 10⁵ (PeCa) or 3 × 10⁵ (C33a) cells were seeded one day prior to transfection into a 12-well plate. The transfection with the M50 or M51 and pEYFP-C1 plasmid was performed using Lipofectamine LTX with Plus reagent. After 24 h the cells were stimulated with Wnt3a-conditioned medium and additionally RSPO1-conditioned medium. 30 mM LiCl served as a positive control. The cells were harvested, transfection efficacy evaluated by flow cytometry (32 +/- 5%), and luciferase as well as Bradford assay conducted as previously described [33]. The luciferase activity was normalized to the protein concentration detected by Bradford assay.