Fig 2: IRF6 and GRHL3 expression in healthy human adult tissues and cells (A) QPCR analysis of IRF6 and GRHL3 in a panel of healthy human adult tissues. Color code refers to tissue groups introduced in Supplementary Figure 1A . (B) IHC for IRF6 as well as a representative H&E staining of a set of normal human tissues. Note that IRF6 exhibits nuclear as well as cytoplasmic localization. Scale Bar: 50 µm. (C) QPCR analysis of IRF6 and GRHL3 in a panel of healthy human cell strains/lines. not detectable (cT > 32) (D) Immunoblot for the proteins IRF6 and E-Cadherin in a set of human cell strains/lines. Note the typical IRF6 double-band indicative of its phosphorylated and non-phosphorylated form, respectively. Full-length blots are shown in Supplementary Figure 6 kDa, kilo Daltons.

Fig 3: IRF6 and GRHL3 overexpression impairs cancer cell proliferation and migration (A) Immunoblots for IRF6 and GRHL3 confirming their overexpression. Full-length blots are shown in Supplementary Figure 6 . kDa, kilo Daltons. (B) Cell growth of empty vector (white)-, IRF6 (black)-, and GRHL3 (gray)- transduced T47D (left) and SCC-68 cell lines (right) shows reduced number of cells by the enhanced presence of IRF6 and GRHL3. * p < 0.05 control vs. IRF6 and GRHL3 (C) qPCR analysis of the proliferation marker KI67 in transduced T47D and SCC-68 cell lines. * p < 0.05 control vs. IRF6 and GRHL3. (D) Transduced cells were analyzed for the expression of EMT markers CDH1, CDH2, VIM, SNAIL1, TWIST2. not detectable (cT > 32). (E) E-Cadherin, N-Cadherin, and Vimentin were analyzed by immunoblots in control and IRF6 and GRHL3 transduced SCC-68 cells. Quantification of the blots is shown below the immunoblots. * p < 0.05 control vs. IRF6 and GRHL3 for N-Cadherin and Vimentin. Full-length blots are shown in Supplementary Figure 6 . kDa, kilo Daltons. (F) Representative live imaging pictures at t=0 h (left) and t=10 h (right) after scratching SCC68 control, SCC68/IRF6, and SCC68/GRHL3. White lanes indicate the migrating fronts on each side of the cell-free gap (left). Quantification of the closure of the cell-free gap is shown on the right. Note a significantly reduced migratory behavior of SCC-68/IRF6 and SCC-68/GRHL3 compared to control starting from t=2 h * p < 0.05 control vs. IRF6 and GRHL3. Scale bar: 500 µm. Movies of the various cell lines are shown in Supplementary Movies 1–3 .

Fig 4: Overexpression of IRF6 and GRHL3 induces differentiation. (A) The differentiation status of three normal cell strains compared to three SCC lines was assessed by qPCR for a panel of differentiation markers (IRF6, GRHL3, IVL, FLG, K10, LOR, and TGM1). The data are shown as a heatmap. Scale: light green – low gene expression; red – high gene expression. (B) IF staining for TGM1, IVL, LOR, and FLG in PA-Ep (non-cancerous) vs. SCC-68 indicates less differentiated cells in the SCC-68 cultures. Scale bar: 50 µm. Note that staining for LOR and TGM1 resulted in a nuclear background staining ( Supplementary Figure 5 ). (C) The same healthy cell strains and SCC cell lines were analyzed in low density (LD) and their corresponding high density (HD) cultures, and the same set of differentiation genes was analyzed by qPCR. The results are shown as heatmap of the fold inductions (HD vs. LD). Scale: light green – low gene induction; red – high gene induction. (D) The effect of forced expression of IRF6 (black) and GRHL3 (gray) compared to control SCC-68 (white) on differentiation markers (K10, FLG, and LOR) was determined by qPCR. * p < 0.05 control vs. IRF6 and GRHL3. (E) Brightfield pictures and corresponding pictures with actin staining indicate the presence of differentiating cell groups (dashed yellow line in the brightfield images and asterisks in the actin staining) in SCC68 cells transduced with IRF6 or GRHL3. Scale bar: 50 µm. (F) K10 staining in dense cultures of transduced SCC-68 cells (left) and its quantification (right). Note the presence of significantly more K10-positive cells in SCC-68/IRF6 and SCC-68/GRHL3 compared to SCC-68 control. Scale bar: 50 µm. * p < 0.05 control vs. IRF6 and GRHL3.