Figure 3: Gain of mesenchymal features by epithelial cells in mouse abdominal and ear skin tissues treated with shikonin. (a) At 24 h after treatment, (1) acetone-, (2) shikonin-, and (3) TPA-treated abdominal skin tissues were stained for the presence of E-cadherin (red), FSP1 (green and indicated with arrowheads), and DAPI (blue) to reveal the appearance of mesenchymal cells in epidermis. The presence of vimentin (red) and FSP1 (green) double-stained cells (yellow and indicated with arrowheads) further indicates that these epithelial cells underwent the EMT process in test skin tissues (4–6). (b) Analogous analysis was performed on treated ear epidermis (7–12). (c) Statistical comparison of EMT activity was performed by scoring the number of yellow fluorescence spots (vimentin+/FSP1+) in (a) and (b). Values shown are the mean ± SEM of three experiments. The significance of differences was analyzed by one way ANOVA ( , , and ).