Fig 2: Viability of mouse meibomian gland explants during different culture time. (A) MTT assay comparison of tissue viability in explants with different culture time (n = 4 in each group). The relative OD values of explants (each performed in triplicate) at 24 hours, 48 hours, 72 hours, and 120 hours compared to that of fresh tissues (0 hour) assessed by MTT assay are shown. (B–F) Morphology of fresh tissues (B) and explants at 24 hours (C), 48 hours (D), 72 hours (E), and 120 hours (F) by H&E staining. (G–K) Immunofluorescence of Krt14 (green), with nuclei labeled by DAPI (blue) in fresh tissues (G) and explants at 24 hours (H), 48 hours (I), 72 hours (J), and 120 hours (K). *Significantly different from the control group (P < 0.05).

Fig 3: Epidermal stem cell (EpSC)-derived extracellular vesicles mediate EpSC development. a Transmission electron microscopy (TEM) and western blot analysis of EpSC-derived extracellular vesicles. b Schematic of percentage of cell components in EpSC-derived extracellular vesicles. Tissue expression (c) and biological process (d) analysis of EpSC-derived extracellular vesicles. e Schematic of cellular component and biological process in spatial function of the epidermis. The depth of the color indicates protein abundance according to log2 normalized protein intensity of EpSC-derived extracellular vesicles. f Interaction network between ITGB1 and other proteins of EpSC-derived extracellular vesicles. g Selected cellular pathways of hemidesmosome and focal adhesion. h Western blot analysis of KRT10, KRT14, ITGB1, ITGA6, ERK1, ICAM1, and ECAD in EpSCs and EpSC-derived vesicles. All biological experiments were repeated three times (n = 3, biological replicates)

Fig 4: 6C can suppress the occurrence of EMT. (A) Immunofluorescence staining of EMT-related genes after 1 week of cell culture. ZEB1 and Snail are transcription factors of EMT, ß-catenin and a-SMA are markers of EMT (green). Nuclei were stained with DAPI (blue). The expression of EMT-related genes in group 6C was significantly reduced. (B) qRT-PCR analysis of ZEB1/2, Snail, ß-catenin and a-SMA gene expression (normalized to ctrl) after 1 week of cell culture. (C) Comparison of cell morphology. a, Treat with 6C for 1 week. b, Treat with 6C for 3 days, then remove 6C and continue to cultivate for 4 days. (D) Comparison of cell morphology. a’, It was first treated with 6C for 3 days, and then cultured with 6C plus 10% FBS for another 4 days. b’, Treat with 6C for 3 days, then remove 6C and continue to incubate with 10% FBS for 4 days. (E) Statistical analysis of cell diameter after C and D treatment. (F) After C treatment, qRT-PCR analysis of P63, K14, K12, Pax6, Ki67, and a-SMA gene expression (normalized to 6C). (G) After D treatment, qRT-PCR analysis of P63, K14, K12, Pax6, Ki67, and a-SMA gene expression (normalized to 6C plus 10% FBS). Data expressed as the means ± SEM from three separate experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no significance).

Fig 5: 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.