Fig 1: Downregulation of FOXP4 promotes squamous differentiation in HaCaT cells. In the recommended maintaining culture condition (1.8 mM CaCl2), intranuclear localization of FOXP4 in HaCaT cells was verified using immunofluorescence staining (A, middle panel). Under calcium-free culture conditions, the intranuclear expression of FOXP4 in HaCaT cells was increased (A, lower panel). (B) mRNA expression of FOXP4 is reduced under the maintaining culture condition (1.8 mM CaCl2) along with increase in IVL expression. (C) mRNA expression of FOXP4 is attenuated by treatment with shRNA for FOXP4 (FOXP4-shRNA3 and 4). (D) Western blot analysis confirmed the reduction in FOXP4 protein expression. (E) RT-qPCR analysis demonstrated that the mRNA expression of KRT1 and KRT10 was significantly increased in FOXP4-shRNA-treated HaCaT cells. Three separate experiments were performed and bars represent SD. The data were analyzed using ANOVA followed by Dunnett test. *p < 0.05; **p < 0.01; N.S., not significant. Scale bars, 50 µm
Fig 2: Downregulation of FOXP4 promotes squamous differentiation in W12 cells. FOXP4-shRNA4 treatment increased mRNA (A) and protein (B) expression of KRT10 and/or IVL. (C) A raft culture of W12 cells. (C-a) Control cells reproduced the whole layers of CIN lesions and squamous-like cells (arrows) in the uppermost layer. (C-b) FOXP4-shRNA4-treated cells developed into a few layers of flattened cells (arrows), which mimic the superficial layer of CIN lesions. KRT10 (C-c) and IVL (C-e) expression were low or moderate in the squamous-like cells (arrows) in control cells. High expression of KRT10 (C-d) and IVL (C-f) is detected in the stratified flattened cells (arrows) in FOXP4-shRNA4-treated cells. FOXP4 expression was suppressed in W12 cells treated with FOXP4-shRNA (C-g and C-h). Three separate experiments were performed and bars represent SD. The data were analyzed using paired t-test. *p < 0.05; **p < 0.01; N.S., not significant. Scale bars, 50 µm
Fig 3: Downregulation of ELF3 recovered FOXP4-induced inhibition of squamous differentiation in W12 cells. (A) FOXP4-shRNA4 treatment altered gene expression of squamous differentiation-related genes. The mRNA expression of TGM1, SPRR1, ELF3, GRHL3, HES2, HES5, NOTCH3, and slightly NOTCH1 and NOTCH2, but not NOTCH4, was significantly promoted. Knockdown of ELF3 by siRNAs inhibited morphological changes in squamous differentiation in FOXP4-shRNA-treated W12 cells (B) and the expression of TGM1, SPRR1, IVL, and GRHL3 as well as NOTCH3, but not NOTCH1 (C). Three separate experiments were performed and bars represent SD. The data were analyzed using ANOVA followed using Dunnett test. *p < 0.05; **p < 0.01; N.S., not significant. Scale bars, 100 µm
Fig 4: Morphological and gene expression changes by downregulation of FOXP4 in W12 cells. (A) Control W12 cells were oval shaped, whereas FOXP4-shRNAs-treated cells show the growth of islands that contain flattened cells (arrows) and stratified structures (arrowheads) with reduction of FOXP4 expression. (B) Stratified structures (arrowheads) were analyzed using sagittal images. (C) The calculated areas of overlapping nuclei (arrows in B) are significantly larger in FOXP4-shRNAs-treated cells. (D) Heatmap of the microarray shows enhanced epithelial differentiation-related genes in FOXP4-shRNA-treated cells. The results are shown as means of individual experiments (N = 3) and bars represent SEM. The data were analyzed using ANOVA followed by Dunnett test. *p < 0.05; **p < 0.01; N.S., not significant. Scale bars, 50 µm (A); and 100 µm (B)
Fig 5: Gene regulatory networks determine dental mesenchymal lineages development during mouse molar formation.a Schematic drawing of a regulon, TFs, transcription factors, T, target gene. b–d Heatmap of cell type-specific regulons and selected top enriched regulons in the cell populations of the dental mesenchyme at E14.5, E16.5 and P3.5. e, f Feature plot and immunostaining of Foxp4 in the mouse molar at P3.5 (n = 3). g–i CT scanning and quantitative analysis of the tooth root length in Foxp4fl/fl and Osr2-Cre;Foxp4fl/fl molars at P16.5, ns, no significant difference. Data are presented as mean values ± SD. n = 4 biologically independent samples, two-tailed t test. j-o H&E staining and immunostaining of periostin in the molars of Foxp4fl/fl and Osr2-Cre;Foxp4fl/fl mice at P16.5 (n = 3). Black and white dotted lines outline the PDL in the mouse molar. The white arrows point to the positive signals and the asterisks indicate absence of the signal. Scale bars in CT images are 200 µm, in other images 100 µm.
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