Fig. 4

Both RNF43 and RNF43ΔPA rescue phenotypes of RNF43/ZNRF3 dKOs cells. a. pcDNA4 plasmids encoding RNF43 wt, RNF43ΔPA and enzymatically inactive RNF43 Mut1 were transfected into CRISPR/Cas9 derived RNF43/ZNRF3 dKO #1 and #2 T-Rex cell lines. Monoclonal stable TetON cell lines were derived thanks to antibiotic selection and colonies picking. b., c. Top flash dual luciferase assay in the RNF43/ZNRF3 dKO #1 (b) and #2 (c) derivates expressing RNF43 wt, RNF43ΔPA or RNF43 Mut1 in the tetracycline sensitive way. Cells in all conditions were treated with the LGK-974. Tetracycline induced expression of RNF43 and its variants, recombinant WNT3A (80 ng/ml) activated canonical Wnt signaling and rRSPO1 (100 ng/ml) co-treatment was used to antagonize RNF43 action. Results were normalized to the untreated samples values and compared using unpaired t-test *p < 0.05, **p < 0.01 N = 3 or 4. d., d’., d”., d”’, e., e’., e”. e”’. Top flash samples were also analyzed by the Western blot for further investigation of the Wnt signaling activity in presence of different RNF43 variants in the RNF43/ZNRF3 dKO #1 (d) and dKO #2 (e). Tetracycline forced expression of both RNF43 wt and RNF43 RNF43ΔPA, but not RNF43 Mut1, suppressed LRP6 1490S, DVL2 and DVL3 phosphorylation events. Recombinant RSPO1 treatment antagonized RNF43 wt, but not RNF43 lacking PA domain. RNF43 lacking enzymatic activity (Mut1) had no effect on the canonical Wnt signaling. DVL2 (d’, e’), DVL3 (d”, e”) and LRP6 (d”’, e”’) activation was quantified by the ImageJ software and presented as ratios of phosphorylation specific upper band to the lower one (arrowheads). Results were normalized to the untreated samples values and compared by t-test, N = 3 *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001