Fig. 3

Preparation of cells lacking functional RNF43/ZNRF3 proteins. a. In order to generate double knockout lines, CRISPR/Cas9 method was applied to introduce mutations in the RNF43 and ZNRF3 genes of T-REx 293 cells. Sequencing results of loci targeted by the CRISPR/Cas9 approach. PAM sequences are underlined. Modifications effects on the protein sequence were predicted. b.RNF43/ZNRF3 dKO cells had higher β-catenin dependent transcriptional activity in response to the overnight incubation with different rWNT3A doses (40, 60 and 80 ng/ml). Also, dKO cell line was insensitive to the 25 ng/ml rRSPO1 treatment. All values are normalized to the unstimulated control cells results. N = 5 (wild type cells), N = 3 (dKO), unpaired two-tailed t-test *p < 0.05, **p < 0.01, *** < 0.001. c. Western blot analysis of canonical Wnt pathway activation in response to the increasing doses of rWNT3A (40, 60 and 100 ng/ml) after 3 h long treatments. RNF43/ZNRF3 dKO exhibited stronger response to the rWNT3A in comparison to the parental cells and rRSPO1 stimulation had no effect. Signal corresponding to the β-actin signal was used as loading control, N = 3