Figure 2. Topical Selectively Apigenin Stimulates Filaggrin Expression in Normal Mouse Epidermis and Keratinocyte Cultures.

5µm paraffin sections were incubated with respective antibodies. The sections were visualized with a Zeiss microscope. Fig. 2a and d are filaggrin staining; b and e are involucrin staining; c and f are loricrin staining. Fig. 2a, b and c are vehicle-treated samples and d, e and f are apigenin-treated samples. Magnifications are the same for all figures. Magnification bars represent 20 µm (a–f).

For Western blot analysis in vivo, differentiation-related protein from mouse epidermis was isolated and quantitated by scanning densitometry, as described in materials and methods. (N=5 for each group). For Western blot analysis in human keratinocyte cultures, Second-passage human keratinocytes isolated from newborn foreskins were cultured in serum-free keratinocyte growth medium containing 0.07 mM calcium. Cells at 80%–90% confluence were switched to medium containing 1.2 mM calcium and treated with either 10 µM apigenin or vehicle alone (0.05% ethanol). After 24 and 48 hours of treatment, keratinocytes were collected for Western blotting. The corresponding protein bands were detected by enhanced chemiluminescence and quantitated by scanning densitometry. Fig 2g & h: displays the quantitative changes of expression in mouse epidermis and human keratinocyte cultures, respectively. Results were presented as percentage of vehicle-treated control, setting vehicle-treated as 100% as indicated by dotted line. Significances are indicated in the figures.