Figure 8.

GRHL3 downregulation promotes a more invasive phenotype of RT4 cells in porcine organ culture. (A) Downregulation of GRHL3 protein by shRNAs in RT4 cell lines is shown by Western blot. Two shRNA constructs targeting GRHL3 and one control shRNA (sh_eGFP) were used to infect RT4 cells. Both shRNA constructs downregulated GRHL3 expression. FOXA1 expression is slightly reduced in RT4 cells after knockdown of GRHL3. (B) Immunolabeling of FOXA1 in RT4 cells grown in organ culture shows that FOXA1 expression can be detected in parental RT4 and in GRHL3 knockdown RT4 cells. Morphologically, parental and shRNA control-transduced RT4 cells formed a multi-layered epithelial lining, while knockdown of GRHL3 by both shRNAs showed more loose cell clusters with beginning stromal invasion (lower panel). Vacuoles in shRNA control-transduced RT4 cells may be indicative of cell stress. Scale bar, 50 µm.