Fig 2: Functional analysis of COL4A6 knockdown in vitro. hGECs transfected with siRNA targeting COL4A6 gene were seeded in the ThinCert cell culture inserts (3D culture), and total RNA and cell lysates were collected after 3 days. mRNA expression levels of COL4A6 (A), KRT1 (B), KRT10 (C), INV (D), KRT13 (E), KRT5 (F), KRT14 (G), KRT15 (H) and KRT16 (I) were measured by real time RT-PCR. The expression of each gene was normalized to that of S29 ribosomal RNA. Bars represent the mean values and standard deviation (+/-SD) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference (Student’s t-tests). Western blot analysis for KRT10 is shown in (J). Full length of western blot data is shown in Supplemental Fig. 7. Results are representative of at least three independent experiments.

Fig 3: Comparison of palatal and buccal mucosa. Histological analysis of oral mucosa in 8-weeks-old mice. HE staining (A) and IHC staining for KRT10 (green) (B) or KRT14 (C) was performed using coronal sections of mouse head. Nuclei were counterstained with DAPI (blue). Boxes indicate the area shown at higher magnification in the right panels. E, epithelial tissue; M, Mesenchymal tissue; T, Tongue; B, bone. Note that KRT10 is highly expressed in keratinized mucosa, whereas KRT14 is expressed in basal cells of both keratinized and non-keratinized mucosa. (D) mRNA expression levels of Krt1 and Krt10 in palatal and buccal mocosa was measured by real time RT-PCR. The expression of each gene was normalized to that of S29 ribosomal RNA. Bars represent the mean values and standard deviation (+/-SD) (n = 3). ***p < 0.001 (Student’s t-tests). Results are representative data of at least three independent experiments. (E) TEM image of palatal mucosa (left) and buccal mucosa (right). Boxes indicate the area shown at higher magnification. Arrows indicate keratohyalin granules. C, stratum corneum; G, Granular layer; S, Superficial layer; I, Intermediate layer.