Fig. 6
Cross-regulation of YAP, Rac1, and Merlin in epithelial layers on NRA. a Immunofluorescence staining for Merlin in epithelial cell sheets on a flat surface and NRA. In the marginal zone and FLPs of sheets on NRA, Merlin was primarily localized in the cytosol (brown boxes), whereas it was localized in the cell–cell contacts of submarginal cells cultured on NRA and flat substrata (red boxes). The samples were fixed after 8 h to remove stencils. b Cells on flat surfaces and NRA were used for Co-IP analysis with the AMOT antibody for investigating physical bounding between AMOT and a target protein, Merlin, and the precipitates were immunoblotted using Merlin antibody. c Lysates from control and YAPKD cells were used for co-immunoprecipitation (Co-IP) study using the AMOT antibody, analyzed by immunoblotting with Merlin antibody. d Control and MerlinKD cells were immunoblotted for evaluation of Rac1 activity via pull-down assay, YAP expression, and its phosphorylation. (n = 3 biologically independent samples, * = statistical significance of the ratio of active Rac1 to Rac1 on control vs. MerlinKD cells. ***P < 5 × 10−3). e Cell migration speed of individual cells in MerlinKD epithelial cell sheets as a function of the distance from the sheet edge in cell sheets cultured on NRA in the presence of a Rac1 inhibitor, NSC23766. (each number of independently analyzed cells, n, is indicated, # = statistical significance of speed values in the marginal region vs. the most submarginal region of MerlinKD cells, ###P < 5 × 10−6. * = statistical significance of control vs. MerlinKD cells without drugs (blue) and of MerlinKD cells with vs. without drugs (black), *P < 0.05 and **P < 5 × 10−3). f Dissemination of cells in control and MerlinKD epithelial sheets on NRA. All error bars are S.E.M. (n = 4 biologically independent experiments, * = statistical significance of the number of disseminations of cells from control vs. MerlinKD epithelial sheets, *P < 0.05). g Schematic of YAP-mediated signaling cascades operating via Rac1 and Merlin, controlling cell speed in cells undergoing partial and complete EMT. All error bars are S.E.M and statistical significance was determined by two-sided Student’s t-test
