Fig. 8.

Peptides disrupting FAM83A-β-catenin interaction inhibit Wnt/β-catenin signaling. a Identification of β-catenin binding α-helical peptide(s) in FAM83A DUF1669 (140aa–210aa) region. The secondary structure of the FAM83A DUF1669 (140aa–210aa) region was analyzed based on the PDB data (4urj). b The FaPC, FaP1, FaP2, and FaP3 fragments were constructed into pEGFP-C1 expression vector connection with a flexible linker ((GGGGS)n). The interactions between β-catenin and the four fragments were evaluated with Co-IP assays in HEK293T cells (n = 3). c, d The predicted docking model of FaP2, FaP3 with β-catenin 530–666 aa region. e, f Kinetic interactions of cell-penetrating peptide TAT-linked peptides with β-catenin was determined by surface plasmon resonance analyses. g Effect of CP-FaP2 and CP-FaP3 on β-catenin-FAM83A and β-catenin-TCF4 interactions in PANC-1 cells. Cells were treated with control (CP-FaPC), CP-FaP2, and CP-FaP3 (5 μM) for 24 hours. Cell lysates were used for IP and western blotting with the indicated antibodies (n = 3). h The schematic diagram of 7TGC plasmid construction and the fluorescence images of PANC-1 cells with control, CP-FaP2, and CP-FaP3 treatment. The relative ratio of green/red fluorescence intensity was quantified (n = 9). i, j Protein and mRNA levels of Wnt target genes AXIN2, C-myc, and CyclinD1 in PANC-1 cells with control, CP-FaP2, and CP-FaP3 treatment (n = 3). k The level of β-catenin ubiquitination in PANC-1 cells after control, CP-FaP2 and CP-FaP3 treatment in PANC-1 cells (n = 3). Cell lysates were used for IP and western blotting with the indicated antibodies. l–n The interaction between β-catenin and AXIN1, GSK3β in PANC-1 cells after control, CP-FaP2 and CP-FaP3 treatment or GFP-tagged FaP1, FaP2, and FaP3 transfection in PANC-1 cells (n = 3). Cell lysates were used for IP and western blotting with the indicated antibodies. o The level of β-catenin ubiquitination in HEK293T cells after GFP-tagged FaP1, FaP2, and FaP3 transfection (n = 3). Cell lysates were used for IP and western blotting with the indicated antibodies. p–r The stability of β-catenin protein upon CHX treatment for indicated times in PANC-1 cells after control, CP-FaP2 and CP-FaP3 treatment or GFP-tagged FaP1, FaP2, and FaP3 transfection (n = 3). Cell lysates were used for IP and western blotting with the indicated antibodies