Journal: Frontiers in Oncology

Article Title: Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas

doi: 10.3389/fonc.2022.1023072

Figure Lengend Snippet: 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.

Article Snippet: Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA).

Techniques: Expressing, Staining, Western Blot

Journal: Frontiers in Oncology

Article Title: Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas

doi: 10.3389/fonc.2022.1023072

Figure Lengend Snippet: IRF6 and GRHL3 downregulation in cancer cell lines (A) Panels of cancer cell lines and corresponding normal cells (striped boxes) were screened for IRF6 , GRHL3 , and CDH1 . Note that RNA was extracted from all cell cultures at a cell density of approximately 70% confluency kDa, kilo Daltons. * p < 0.05 control vs. cancer cell lines; # not detectable (c T > 32). HME: human melanocytes. (B) Immunoblots for IRF6 and E-Cadherin in a subset of the analyzed cancer cell lines and controls. Full-length blots are shown in Supplementary Figure 6 . HME: human melanocytes. (C) Brightfield (BF) pictures, IRF6 staining, as well as the merge of IRF6 (green) with actin (red) and cell nuclei (blue) is shown for breast and lung cancer, skin and oral SCC samples and their corresponding normal controls. Note that IRF6 is predominantly expressed in the cytoplasm of the various cells. Scale bar: 25 µm.

Article Snippet: Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA).

Techniques: Control, Western Blot, Staining

Journal: Frontiers in Oncology

Article Title: Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas

doi: 10.3389/fonc.2022.1023072

Figure Lengend Snippet: 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 .

Article Snippet: Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA).

Techniques: Over Expression, Migration, Western Blot, Plasmid Preparation, Control, Marker, Expressing, Imaging

Journal: Frontiers in Oncology

Article Title: Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas

doi: 10.3389/fonc.2022.1023072

Figure Lengend Snippet: 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.

Article Snippet: Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA).

Techniques: Over Expression, Gene Expression, Staining, Expressing, Control, Transduction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) Comparison of the colony morphology, Keratin10 ( KRT10 ) and IRF6 expression in three individual primary oral mucosa-derived keratinocytes (Mucosa 1-3) compared to three foreskin-derived keratinocytes (Skin A-C). Note that the skin-derived cells form tightly packed colonies, express high levels of KRT10 , and express more IRF6 than mucosa-derived cells. ∗ p < 0.05 (IRF6 and KRT10 levels in Skin vs. Mucosa). Scale bar: 100 μm. kDa: kilo Dalton. (B) KRT10 and IRF6 mRNA levels of mucosal- and skin-derived keratinocytes from the same donor show higher expression of both genes in the skin. ∗ p < 0.05 ( KRT10 and IRF6 levels in Skin vs. Mucosa). (C) Venn diagram of the proteomic analysis comparing OKF6/TERT2 (oral mucosa) and N/TERT1 (foreskin) keratinocytes reveals a high number of proteins present in both cell lines, 189 and 159 proteins are unique to OKF6/TERT2 and N/TERT1, respectively. From the 3,036 shared proteins, 91 proteins are differentially expressed (>2 fold) and mostly associated with the biological processes of epidermal differentiation, keratinization, and cornification (table right). (D) Similar to the observations in primary keratinocytes, N/TERT1 keratinocytes form more cohesive and regularly shaped colonies and express higher levels of KRT10 and IRF6 than the oral mucosa-derived OKF6/TERT2. Scale bar: 100 μm. ∗ p < 0.05 ( KRT10 levels in Skin vs. Mucosa); ∗ p < 0.05 (IRF6 levels in Skin vs. Mucosa). Full-length immunoblots are shown in .

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Comparison, Expressing, Derivative Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) In the absence of IRF6, OKF6/TERT2 and N/TERT1 keratinocytes change their colony morphology as assessed by live imaging, F-actin (phalloidin, red), E-Cadherin (green), and crystal violet (CV) staining. DAPI was used to stain for nuclei (blue). Scale bars: 20 μm (Live Imaging); 150 μm (F-Actin, E-Cadherin); 100 μm (CV). (B) IRF6 knockout in both OKF6/TERT2 and N/TERT1 cell lines results in significantly more single cells (sc), reduced colony circularity (cc) as well as in the emergence of larger single cells (cell area) when compared to their respective controls. ∗ p < 0.05 controls vs. clones (sc); ∗ p < 0.05 controls vs. clones (cc, cell area). (C) Immunoblots show overexpression of IRF6 and GRHL3 in the IRF6-deficient keratinocyte clones after transduction. Note the very little expression of ectopic IRF6 in N/TERT1 IRF6 knockout clones. kDa: kilo Dalton; ex.: exogenous. (D) Live imaging and crystal violet pictures of typical colony morphologies upon re-expression of IRF6 in the IRF6 K.O. clones. Note that low IRF6 levels are not able to rescue the dispersed colony phenotype. Scale bar: 100 μm. Full-length immunoblots are shown in .

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Imaging, Staining, Knock-Out, Clone Assay, Western Blot, Over Expression, Transduction, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (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 .

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Control, Clone Assay, Western Blot, Staining, Knock-Out

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) Live Imaging pictures of the in vitro scratch at the indicated times after wounding the confluent monolayer and before scratching. Left side: OKF6/TERT2; Right side: N/TERT1. Scale bar: 500 μm. (B) Quantification of the manual scratch shows a delay of the wound closure in the absence of IRF6. ∗ p < 0.05 control vs. clones. (C) Quantification of an automated live imaging scratch assay confirms impaired closure of the scratch in the IRF6 knockout keratinocytes compared to control. ∗ p < 0.05 control vs. clones. (D) Pictures of N/TERT1 control and N/TERT1 clone #10 taken at the time of scratching (0 h), 5 h, and 10 h shows that lack of IRF6 results in significantly more cells that move randomly as single cells (arrowheads, right side) compared to control (left side).

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Imaging, In Vitro, Control, Clone Assay, Wound Healing Assay, Knock-Out

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) Live Imaging pictures and F-actin staining (phalloidin, red) with or without (only Live Imaging) exogenous Ca 2+ addition. While the control keratinocytes start to differentiate (asterisk and arrowheads), lack of IRF6 impairs differentiation. Scale bar: 200 μm (Live Imaging); Scale bar: 150 μm (F-actin). (B) Heatmaps of the qPCR analyses of various differentiation markers in basal (0.1 mM) vs. high Ca 2+ (1.2 mM) conditions. Note that in the absence of IRF6 all the differentiation markers are not induced upon the Ca 2+ -switch. n.d.: not detectable (Ct > 32); ctrl.: control. (C) qPCR analyses of specific differentiation markers showing the lack of induction upon the addition of Ca 2+ in the IRF6 knockout clones compared to controls. ∗ p < 0.05 basal vs. 1.2 mM Ca 2+ . ctrl.: control. (D) Immunofluorescent staining for the markers Involucrin (IVL, green) and Transglutaminase 1 (TGM1, green) in OKF6/TERT2 cells and for IVL (green) and Loricrin (LOR, green) in N/TERT1 keratinocytes. Note that all differentiation markers were robustly induced in the control cells in the presence of IRF6. Scale bar: 50 μm. DAPI was used to counterstain the cell nuclei (blue).

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Imaging, Staining, Control, Knock-Out, Clone Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) H&E staining of 3D skin organotypic cultures obtained with N/TERT1 control and N/TERT1 clones #8 and #10 keratinocytes. The various skin layers are indicated. SB: stratum basale; SS: stratum spinosum; SG: stratum granulosum; SL: stratum lucidum; SC: stratum corneum. Scale bar: 100 μm. (B) Loricrin is strongly expressed in organotypic cultures of control keratinocytes in the stratum granulosum. In contrast, Loricrin is only weakly and irregularly expressed in the absence of IRF6. Close-up images are shown to the right of each organotypic culture. Arrowheads indicate aberrant presence of nuclei in the SC. (C) Quantification of H&E pictures of the 3D skin models. Note that in the absence of IRF6 the area of the SL + SC (orange area left panel), as well as the SG (orange area middle panel) are significantly reduced. Also, absence of IRF6 results in the presence of cell nuclei (arrowhead) in the SL and SC, which is not the case for control (right panel). ∗ p < 0.05 control vs. clones #8 and #10.

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Staining, Control, Clone Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes

doi: 10.3389/fcell.2021.718066

Figure Lengend Snippet: (A) Heat map of hierarchical clustering after imputation and color key represent the total amount of identified proteins in N/TERT1 (left side) and OKF6/TERT2 samples (right side). Note the hierarchical clusters on top demonstrating significant changes in the proteome between skin- and oral mucosa-derived cell lines. (B) Differentially expressed proteins in OKF6 clone #26 vs. OKF6 control in cultures at high cell density (HD). Tables on the top summarize the main three protein clusters: immune response/IFN-related (green), Skin Homeostasis (yellow), and ECM (red). String network depicting all differentially expressed proteins is shown as well with the three main clusters as described before. Note that only proteins are shown having a differential expression of at least log 2 fold change = 1. ECM: extracellular matrix. (C) Differentially expressed proteins in N clone #10 vs. N control in cultures at high cell density (HD). Tables on the left summarize the main three protein clusters: immune response/IFN-related (green), Skin Homeostasis (yellow), and ECM (red). String network depicting all differentially expressed proteins is shown as well with the three main clusters as described before. Note that only proteins are shown having a differential expression of at least log 2 fold change = 1. ECM: extracellular matrix. (D) qPCR verifications for some mRNA transcripts ( GAL7 , S100A8 , S100A9 , DSG1 , and DSC2 ) for which the corresponding proteins have been found to be differentially expressed in both OKF6 and N cells compared to the corresponding IRF6-deficient keratinocytes. ∗ p < 0.05 controls vs. clones #15 and #26, and clones #8 and #10.

Article Snippet: Primary antibodies used for immunofluorescent staining: Rabbit polyclonal antibodies anti-IRF6 (NBP2-49383, Novus Biologicals, Centennial, CO, United States), anti-E-Cadherin (20874-1-AP, Proteintech), anti-TGM1 (NBP2-34062, Novus Biologicals), and anti-Loricrin (PA5-30583, Thermo Fisher Scientific).

Techniques: Derivative Assay, Control, Quantitative Proteomics, Clone Assay

Journal: The Journal of Clinical Investigation

Article Title: A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias

doi: 10.1172/JCI40267

Figure Lengend Snippet: Table 1

Article Snippet: The following antibodies were used: monoclonal antibodies against HA (F-7) and Myc (9E10) epitopes, p63 (4A4), polyclonal antibodies against PCNA (PC10), and actin (C11) were purchased from Santa Cruz Biotechnology Inc.; anti-keratin (anti-K1) was purchased from Covance; mouse polyclonal anti-IRF6 from was purchased from Abnova Inc.; and goat polyclonal anti-IRF6 antibody (IMG3484) was purchased from Imgenex.

Techniques: Expressing