Fig. 7: Testing dominant-negative potential of ZNRF3 variants at endogenous levels.

A Endogenous levels of heterozygous missense mutations do not affect ZNRF3 function more strongly than heterozygous knockout mutations. β-catenin reporter assays for knockin/out ZNRF3 clones. All heterozygous mutations, including P179L, R307W, P331L/H, and WT+frameshift (fs), slightly increase β-catenin signaling (2–8 fold) to similar levels. Homozygous ZNRF3 knockout clones, homozygous R307W or P331H knockin clones, and RNF43/ZNRF3 double knockout clones, all show a comparable further increase in signaling (18–30 fold). The relative Wnt/β-catenin signaling activities are depicted as WRE/CMV-Renilla ratios, in which the value obtained for wild-type (WT) HEK293T cells was arbitrarily set to 1. Wnt3A conditioned medium was added to WT HEK293T cells and all CRISPR-edited HEK293T cells. Statistical significance was assessed using a T-test (****P < 0.0001, **P < 0.01, *P < 0.1). For each heterozygous amino acid variant, 3–4 individual clones were used. Values for each individual clone are shown in Supplementary Fig. 17B. B A β-catenin reporter activity analysis performed for representative supposedly dominant-negative RNF43 or ZNRF3 variants, shows that overexpression of these hyperactive variants reduces β-catenin signaling much weaker than a complete knockout of both proteins. Wnt3A was added to all samples except for control-treated empty vector (EV) transfected cells, serving as negative control. All relative β-catenin reporter activities are depicted as WRE/CMV-Renilla ratios, in which the value obtained for EV with L-Control was arbitrarily set to 1. Statistical significance was assessed using a one-way ANOVA test (****P < 0.0001).