Fig. 2

RNF43ΔPA inhibits canonical Wnt signaling pathway. a. Top Flash dual luciferase assay performed in the T-REx 293, RNF43 TetON and two RNF43ΔPA TetON cell lines in the response to the 100 ng/ml rWNT3A and 50 ng/ml rRSPO1 treatments in the presence of 0.5 μM LGK-974. Both RNF43 variants efficiently inhibited cellular responses. Results were normalized to Tet free conditions. N = 4, unpaired two-tailed t-test ***p < 0.001, ****p < 0.0001. b. Expression of the canonical Wnt signaling target gene AXIN2 in response to the rWNT3A (100 ng/ml) and rRSPO1 (50 ng/ml) treatments. Cells overexpressing RNF43ΔPA mutant, similarly to the wild type RNF43, showed weaker AXIN2 expression. Results were normalized to the assay values of the unstimulated samples. N = 3, unpaired two-tailed t-test. *p < 0.05, **p < 0.01, *** < 0.001. Data are presented as 2^−ΔΔCt ± SD. c. Schematic representation of performed experiments elucidating an impact of RNF43ΔPA on the different canonical Wnt signaling modes of activity – Wnt pathway activation and RNF43 constructs expression simultaneously, before and after pathway stimulation. d. Western blot analysis of RNF43ΔPA impact on the canonical Wnt pathway components. Phosphorylation of S1490 of LRP6 together with the DVL2 and DVL3 activation manifested by electrophoretic, phosphorylation specific shifts (arrowheads) upon RNF43 or RNF43ΔPA tetracycline forced expression along the 80 ng/ml rWNT3A and 25 ng/ml rRSPO1 treatments (d), before them (d’) or after (d”) stimulations. Both variants of RNF43 showed inhibitory effect on the β-catenin dependent Wnt signaling in the all tested conditions. LGK-974 was used to inhibit autocrine production of the Wnt ligands, HA tag signal corresponds to the RNF43 proteins and β-actin was employed as loading control