Fig 1: YAP regulates E-cadherin through WT1 in epithelial layers on NRA. a Immunofluorescence staining for WT1 in YAPKD cell sheets (top), and for YAP in WT1KD cell sheets cultured on NRA. The samples were fixed after 8 h to remove stencils. b Co-IP analysis using the YAP antibody, followed by immunoblotting using the WT1 antibody. c Chromatin immunoprecipitation (ChIP) analysis of the WT1-YAP complex binding at the E-cadherin promoter (n = 3). We extracted cross-linked chromatin and immunoprecipitated it using antibodies against YAP, WT1, IgG (negative control), and RNAPII or histone H3 (see Supplementary Fig. 12a) antibodies (positive controls). The immunoprecipitated chromatin and input genomic DNA were used for amplification of the E-cadherin promoter, CTGF promoter (known to be regulated by YAP but not WT1, and used as another control), and GAPDH promoter (n = 4 biologically independent samples, * = statistical significance of PCR products from each sample vs. IgG, *P < 0.05 and **P < 0.01). d E-cadherin expressions of control and WT1KD cells analyzed by immunoblotting with WT1 and E-cadherin antibodies (n = 3 biologically independent samples, * = statistical significance of E-cadherin expression of control and WT1KD cells, ***P < 5 × 10-3). e Cell migration speed of individual cells in control and WT1KD epithelial cells in the marginal region of the sheets in the presence of an E-cadherin blocking antibody (each number of independently analyzed cells, n, is indicated, * = statistical significance, n.s = no significance, and ***P < 5 × 10-3). f Dissemination of cells in control and WT1KD epithelial sheets on NRA in the presence of an E-cadherin blocking antibody (n = 4 biologically independent experiments, * = statistical significance, *P < 0.05, ***P < 5 × 10-3, and n.s = no significance). g Immunoblotting of phosphorylated YAP and total YAP in cell sheets cultured in the presence of different concentrations of E-cadherin blocking antibody (n = 3 biologically independent samples). h Schematic of the YAP-mediated cell dissemination triggered by mechanical cues stemming from NRA, operating through E-cadherin control by YAP-WT1 complexes and leading to cell dissemination in epithelial cell sheets on NRA. All error bars are S.E.M and statistical significance was determined by two-sided Student’s t-test