Fig 2: FIH1 and K15 Are Direct Targets of miR-184 that Maintain Epidermal Stemness In Vitro(A) HEK293 cells were co-transfected with the indicated concentration (nM) of pre-miR-184 (PM184), disease-causing pre-miR-184C57U mutant (C57U) or pre-miR-203 (PM203), or control oligonucleotides together with luciferase plasmids containing the 3'UTR of the indicated genes downstream to the luciferase encoding sequence. Cells were lysed 24 hr later and the luciferase assay was performed as detailed in the Experimental Procedures.(B and C) In situ hybridization of miR-184 coupled with K15 immunostaining on mouse head cryosections at P0 (B) and P8 (C). Higher magnification pictures of (C) are shown in (i) and (ii).(D) Immunofluorescent staining of K15 on mouse sections of newborn mice of the indicated genotypes. Arrows show abnormal expression of K15 in supra-basal cells of KO mice.(E) Primary KCs of the indicated genotypes were lysed and subjected to western blot analysis for K15, FIH1, or ERK as loading control.(F) KCs were transfected with endoribonuclease-prepared silencing RNAs against K15 (siK15) or FIH1 (siFIH1) or GFP (siGFP) as control. Lysates were prepared 48 hr later for immunoblotting against the indicated proteins.(G and H) Primary human foreskin KCs were transfected with the indicated silencing RNAs and then subjected to the clonogenicity test. Colonies were visualized by Rhodamine staining (G, examples of two experiments are shown), and quantification by computerized analysis is shown in (H).Data shown are means ± SD from three independent experiments. *p < 0.05 statistically significant by Student's t test. Quantification of western blot analysis from at least three independent experiments (p < 0.05) is shown at the bottom of each panel. The dashed line in (B) indicates the dermal-epidermal junction. Scale bars are 50 µm for (B) and (C) and 20 µm for (D). de, dermis; ep, epidermis; Mx, matrix; ORS, outer root sheath.

Fig 3: Investigation of the effects of HFMSCs on DSS-induced UC. (a) The morphology of HFMSCs was observed under a white-light microscope. (b, c) HFMSCs stained with Alizarin red and Oil red O differentiated into osteoblasts and adipocytes. (d) CK15 was observed by immunofluorescence. (e) Surface markers of HFMSCs, including positive markers (CD90 and CD29) and negative markers (CD31 and CD43), were detected by flow cytometry. HFMSCs were administered to DSS-treated mice. (f, g) The colon lengths of the mice in all the groups were measured and analysed. (h, i) The body weight loss and DAI values were compared among the three groups. (j) The colon histology of the mice in the three groups was detected by HE staining. (k) Mucosal damage and inflammatory infiltration in the colons were determined. All the data are displayed as the means ± SDs. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant. The images were obtained with 10x and 20x Olympus dotSlide objectives.

Fig 4: Immunohistochemistry of rabbit eyes. Frozen normal and postoperative eye specimens were used for immunohistochemical examination. The left column shows images of a normal cornea (A–E). (A) Hematoxylin-eosin (HE) staining reveals five to six layers of corneal epithelial cells in the central region. K3 is positive in the cytoplasm of cells in the surface layer of the epithelium (B), while K13 (C), p63 (D), and K15 (E) are negative in all layers. The central column shows images of a normal limbus and conjunctiva (F–K). HE staining of the normal limbus and conjunctiva is displayed in (F,G), respectively. K3 is negative in the normal conjunctiva (H), while K13 shows cytoplasmic positivity in the upper layer of the normal limbus (I). Nuclear p63 positivity is seen in the basal layer of the limbus (J), along with cytoplasmic K15 positivity in basal layer cells (K). The right column shows the central cornea of a SOMET group eye (L–Q). HE staining of the central cornea reveals 3–5 layers of flat epithelial cells (L). HE staining also shows that the transplanted oral mucosal graft consists of stratified squamous epithelium (M). In the deeper layer at the graft site, basal cells with a high N/C ratio exist close to the basal layer (M). In the expanding epithelium, the surface layer of cells demonstrates cytoplasmic K3 positivity (N) and K13 is also positive in the cytoplasm of superficial epithelial cells (O). Basal layer cells at the graft site show nuclear staining for p63 (P) and cytoplasmic staining for K15 (Q). In the Control group, HE staining revealed no epithelial proliferation and multinucleated spheres were attached to the exposed corneal stroma (R). Neither K3 positivity (S) nor K13 positivity (T) was detected in the Control group.

Fig 5: Immunohistochemical staining of oral mucosal epithelium. Submucosal connective tissue was removed by dispase treatment and the superficial tissues were used for immunohistochemistry. Apart from 3 to 5 basal layers, the oral mucosal epithelial cells are diffusely stained by K3 (top left) and K13 (top right), consistent with the phenotype of oral mucosal epithelium. Some basal cells show cytoplasmic positivity for K15 (bottom left), while there is nuclear P63 positivity in the lowest basal cell layer (bottom right).