PMC4943770_01
Slit-lamp images of the left eye of the CIN patient before the 2nd operation (a) and 14 months after the 2nd operation (b). Histological examination of the CIN tissue (c). Arrow heads indicate that nuclei of the superficial cells of the CIN epithelium remained as a shrunken pyknotic structure. H&E staining; bar = 100 mum.
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F1
During the course of several, regular postoperative checkups, a dysplastic lesion recurred, and then gradually enlarged until the lesion covered almost the entire cornea and 360  of the corneal limbus (fig. 1).
Images showing immunostaining results against cytokeratins and keratinization markers. Cytokeratins 1 (a-1, a-2), 3 (b-1, b-2), 4 (c-1, c-2), 6 (d-1, d-2), 8 (e-1, e-2), 10 (f-1, f-2), 12 (g-1, g-2), 13 (h-1, h-2), and 16 (i-1, i-2), and transglutaminase (j-1, j-2), involucrin (k-1, k-2), and filaggrin (l) for the CIN tissue (a-1-k-1) and the normal corneal tissue (a-2-k-2). Bar = 100 mum. The brackets indicate the CIN and corneal epithelial layer. Note that the superficial staining seen in the CIN epithelium is an artifact, because it was also found in the negative control samples.
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F2
CIN epithelium, while all epithelial layers diffusely expressed this cytokeratin in the normal corneal samples (fig. 2).
Images of the immunostaining results. Immunostaining against Ki-67 (a-1, a-2), the results of TUNEL analyses (b-1, b-2), hTERT expression (c-1, c-2), phalloidin (e-1, e-2) and antibodies against E-cadherin (f-1, f-2), alpha-smooth muscle actin (alphaSMA) (g-1, g-2), and N-cadherin (h-1, h-2) for the CIN tissue (a-1-c-1, e-1-h-1) and the normal corneal tissue (a-2-c-2, e-2-h-2). Bar = 100 mum. d Real-time PCR data about the expression of hTERT mRNA. The asterisk means that the signal was below the detection limit. The arrow heads indicate cells positive for N-cadherin, which are thought to be potential corneal epithelial stem cells. HCE-T = Human corneal epithelial large T antigen cells. The CIN epithelium demonstrated a large number of apoptotic cells. The hTERT gene was significantly upregulated in the CIN epithelium in both RNA and protein levels compared to the normal corneal epithelia.
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F3
The expression level was comparable to that measured in the human corneal epithelial cells, the human corneal epithelial large T antigen cells, a cell line which was immortalized by the adenoviral introduction of SV40 large T antigen (fig. 3). In addition, the CIN epithelium did not express the other mesenchymal marker, N-cadherin (fig. 3), while the normal limbal epithelium exhibited the expression of this protein at its basal layer, where stem cells of the corneal epithelial cells reside.