Vimentin

On day 0, 1.0 × 10³ cells per well were seeded in 96-well plates. At the indicated hours, sulforhodamine B (Sigma, St Louis, MO, USA) assay was used to obtain relative estimates of viable cell number as previously described in Piro et al.^{21 (link)} Western blot analyses were carried out as described previously in Melisi et al.^{22 (link)} Briefly, cell lines were washed twice with cold phosphate-buffered saline and lysed at 4 °C into RIPA buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) plus protease inhibitor mix (Sigma-Aldrich). Each lysate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with antibodies against E-cadherin, β-catenin (Abcam, Cambridge, UK), receptor tyrosine kinase-like orphan receptor 2 (ROR2), Histone H3, glycogen synthase kinase 3α/β (GSK3α/β) and p-GSK3β-(S9) (Cell Signaling Technology, Boston, MA, USA), vimentin (Dako, Glostrup, Denmark), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and γ-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive proteins were detected using an enhanced-chemiluminescence reagent (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Images were captured by LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK).

We reassess the histopathological slides of the 42 patients that received diagnosis of OLP based on the criteria proposed by the WHO.^{22 (link)} Histological signs of dysplasia were exclusion criteria. Immunofluorescence was performed as previously reported^{5 (link)} using a double staining with anti acetyl-histone H3 (cell signaling) and vimentin (DAKO, Carpinteria, CA) and anti phospho-histone H2A.X (Millipore, Billerica, CA) as primary antibodies followed by FITC or TRITC-conjugated secondary antibody (Covance, Berkeley, CA). DNA was stained using Hoechst 33342. Images of 4 to 10 fields of each case were captured at 400× magnification using a QImaging-ExiAqua monochrome digital camera attached to a Nikon Microscope (Nikon, Melville, NY) and visualized with QCapturePro 7 software (Surrey, BC, Canada). We performed morphometric image analysis for γH2AX using the software ImageJ (Version 1.38s; NIH, Bethesda, MD). All positive and negative cells were counted in each field and the percentage of total number of cells in each case was calculated. For ac.H3K9ac staining intensity analysis, all cases were classified as negative (0), weak (+), moderate (++), or strong (+++). The intensity of H3K9ac was graduated independently by 3 oral pathologists.

GE were fixed in 4% paraformaldehyde and processed for paraffin embedment. After rehydration, tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for histological examination or processed for immunohistochemistry (IHC) to study expression of specific proteins. For IHC, after antigen retrieval, sections were incubated for 1 h with mouse antibodies against human involucrin (Novocastra), K10 (Santa Cruz Biotechnology), K13 (Monosan), vimentin (Dako), or Ki-67 (Dako) as previously described.^{3 (link),4 (link)} Collagen type IV (Monosan) and laminin 5 (Dako) IHC were performed on cryosections without antigen retrieval. Hereafter, the sections were washed in PBS and incubated for 30 min with Envision (Dako), except for K13, which was incubated with PowerVision Poly-HRP (Leica Biosystems) for one hour. Next, the sections were incubated with AEC substrate for 10 minutes followed by hematoxylin staining. The microscopic slides were visualized and recorded with a Nikon Eclipse 80i. Contrast enhancement and quantification of reepithelialization on H&E sections were done with NIS-Elements software (Nikon Instruments Europe B.V.).