Fig. 6: Alternative YAP-β-catenin signaling axis.

A Luciferase experiments with MCAT and MCATmut reporter constructs were performed with melanoma cell lines 48 h after siβ-catenin or siCtr transfection. Values were normalized to MCATmut control transfection and siCtr was set as 1 for each cell line (n = 3). Bars are shown as mean ± SEM (multiple T-tests (Bonferroni-Dunn) *P < 0.05, ns: not significant). B, C Flow cytometry analyses with MCAT-eGFP were performed with melanoma cell lines 48 h after siβ-catenin or siCtr transfection. B Analyses of high GFP-staining in melanoma cells. siCtr was set as 1 for each cell line. C Analyses of low GFP-staining in melanoma cells. siCtr was set as 1 for each cell line (left) (n = 3). Bars are shown as mean ± SEM (multiple T-tests (Bonferroni-Dunn) *P < 0.05, ns: not significant). Representative histograms of GFP-staining’s in SBcl2 cells 48 h after siβ-catenin or siCtr transfection. Cells were gated into low (+) and high GFP-staining (+++). D Relative number of cells with nuclear YAP localization 48 h after transfection of siβ-catenin or siCtr visualized with YAP localization plasmid. siCtr was set as 1 (n = 3). Bars are shown as mean ± SEM (multiple T-tests (Bonferroni-Dunn) *P < 0.05, ns: not significant). E Relative number of cells with cytoplasmic YAP localization 48 h after transfection of siβ-catenin or siCtr visualized with YAP localization plasmid. siCtr was set as 1 (n = 3). Bars are shown as mean ± SEM (multiple T-tests (Bonferroni-Dunn) *P < 0.05, ns: not significant). F Representative pictures of SBcl2, WM1366 and MV3 cells stained with DAPI (blue) and YAP (green) (right). G Interaction of YAP and β-catenin measured by co-immunoprecipitation with anti-YAP antibody in SBcl2 cells (lane 2). Lane 1 represents input of SBcl2 cells and lane 3 immunoprecipitation with anti-IgG antibody in SBcl2 cells. Immunoblotting with anti-YAP and anti β-catenin antibodies. H Interaction of TEAD, YAP and β-catenin analyzed by co-immunoprecipitation with anti-TEAD antibody in SBcl2 cells (lane 2). Lane 1 represents input of SBcl2 cells and lane 3 immunoprecipitation with anti-IgG antibody in SBcl2 cells. Immunoblotting with anti-TEAD and anti β-catenin antibodies. I EMSA with γ-³²P-ATP-labeled MCAT consensus sequence (consensus) oligonucleotide containing one predicted TEAD binding sites. Analysis of TEAD-Oligo band was performed with siTEAD (lane 2), anti-YAP antibody (lane 3, 5) and anti-β-catenin antibody (lane 4, 5). Star: TEAD-oligo binding. Arrow: reduction of the TEAD-Oligo band or supershift due to complexing of YAP-β-catenin-TEAD.