Figure 4

Knockdown of YAP attenuates shE2A-induced invasion and migration. A, Downregulation of E2A by shE2A resulted in increased YAP mRNA expression while transfection of plasmids encoding E12 or E47 decreased YAP mRNA in shE2A treated SW480 cells. B, E2A decreased YAP protein expression determined by immunoblot analysis. C, YAP knockdown by shYAP in SW480/shE2A cells suppressed the enhanced cell invasion and migration ability caused by shE2A to a similar level as observed in SW480/shNC cells. D, Downregulation of YAP by shYAP decreased the invasion and migration ability of SW480 cells. Migration/invasion ability of SW480 decreased after shYAP was transfected into SW480 cells. Representative photos of stained cells are shown with the original magnification of 100×. Scale bars: 100 μm. E, Immunoblot analysis of YAP, E-cadherin, vimentin, β-catenin and MMP-9 with GAPDH as loading control. Epithelial marker E-cadherin expression was elevated and mesenchymal marker vimentin and β-catenin expression was reduced after shYAP was transfected into SW480 cells. MMP-9 was decreased after shYAP transfection. Data in the histograms is expressed as the means ± SD from 3 separate experiments. F, Representative images of immunofluorescence analysis of EMT markers. E-cadherin was enhanced while vimentin and β-catenin were decreased after shYAP transfection. Magnification: 400×; Scale bars: 50 μm. *, P < 0.05; **, P < 0.01.