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Figure 2. Differential expression and localization of wit 3.0 mRNA. (A) RNA transfer blot analysis showed that AA1 hybridized the edentulous oral mucosal mRNA sample at the size of 3.0 kb [lane 1] but did not hybridize the untreated gingival sample [lane 2]. GAPDH hybridized equally. (B) Diagrams of wound inducible transcript 3.0 (wit 3.0) and overlapping full-length cDNAs, AA1 (410 bp), CS13 (1980 bp), and CS312 (843 bp). Open reading frame (ORF) was found in the overlapping area of CS13 and CS312. Red arrows indicate primers flanking the ORF region that were used for RT-PCR assay. (C) The actively healing post-extraction oral mucosa adjacent to the extraction socket [S]. Epithelial proliferation and migration and active connective tissue re-organization were observed at the edge of the ruptured gingival square (corresponding to Fig. 2D ) (bar = 200 µm). (D) Arrowheads represent connective tissue fibrosis found next to the infiltrated inflammatory cells [*] adjacent to the extraction socket [S] (bar = 200 µm). (E) Positive in situ hybridization (blue staining, arrows) was seen in the fibroblasts adjacent to the extraction site, where dense fibrosis-like tissue was found adjacent to the extraction socket [S] (bar = 200 µm). (F) Negative sense control in situ hybridization. No hybridization was detected in the area adjacent to the extraction socket [S] (bar = 200 µm). (G) RT-PCR analysis of total RNAs from human oral fibroblast (lane 1), human oral keratinocyte (lane 2), untreated rat gingival fibroblast (lane 3), and edentulous rat oral mucosal fibroblast (lane 4). The strong expression of wit 3.0 was observed only in the cultured fibroblast derived from edentulous oral mucosa. The result also indicated two PCR products with the size of 645 bp (wit 3.0  ) and 764 bp (wit 3.0 ß). Beta actin RT-PCR served as a housekeeping gene control.
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