Fig 2: (A) qPCR analyses for the epithelial markers CDH1, GRHL3, and IRF6 as well as for the mesenchymal markers FN, VIM, and SNAIL in OKF6/TERT2 (left) and N/TERT1 (right) cell lines. Note that although there is a slight increase of the mesenchymal markers, CDH1 does not decrease in the absence of IRF6. ctrl.: control; *p < 0.05 controls vs. clones #15, #26, #8, #10. (B) Immunoblots confirm the qPCR results and show no change in E-Cadherin, but an increase in FN, and absence of IRF6 in the clones. kDa: kilo Dalton; ctrl.: control. (C) Treatment of N/TERT1 controls (top row) with the EMT-inducing growth factors EGF, TGFß1, and TGFß3 for 72 h induces changes in the cellular morphology with scattered and enlarged cells (arrowheads). In N/TERT1 clone #10 (bottom row) a similar morphological phenotype can already be appreciated without addition of growth factors. *indicates enlarged cells. Scale bar: 150 µm. (D) Crystal violet staining and cell area analysis of N/TERT1 control in the absence and presence of TGFß1 (72 h) reveals the emergence of enlarged cells in the presence of the growth factor. *p < 0.05 control vs. TGFß1-treated cells; ctrl.: control. (E) qPCR analyses for the mesenchymal markers FN, VIM, and SNAIL as well as the epithelial markers CDH1, IRF6, and GRHL3 in TGFß1-treated (72 h) cells in the presence (control) or absence (clone #10) of IRF6. Note that the mesenchymal markers as well as IRF6 increase, while CDH1 does not change. Also note that IRF6 is required for a proper modulation of all these markers. #p < 0.05 untreated vs. TGFß1-treated cells; *p < 0.05 control vs. IRF6 knockout cells (clone #10). Full-length immunoblots are shown in Supplementary Figure 9.