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. 2021 Sep 30;9:718066. doi: 10.3389/fcell.2021.718066

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Copyright © 2021 Girousi, Muerner, Parisi, Rihs, von Gunten, Katsaros and Degen.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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(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, TGFβ1, and TGFβ3 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 TGFβ1 (72 h) reveals the emergence of enlarged cells in the presence of the growth factor. ^∗p < 0.05 control vs. TGFβ1-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 TGFβ1-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. TGFβ1-treated cells; ^∗p < 0.05 control vs. IRF6 knockout cells (clone #10). Full-length immunoblots are shown in Supplementary Figure 9.