RASSF9 Regulated Epidermal Homeostasis Figure 8. Aberrant differentiation in the skin of RASSF92/2 mice—K1 (red). (A) Frozen sections of dorsal skins of WT (+/+) and RASSF92/2 (2/2) mice were immunostained with red fluorescence for K1 in 4-dpp mice. The dashed white lines denote the epidermis-dermis border. Similar results were obtained from three independent pairs of mice. Scale bar = 100 mm. Note the abnormal staining of K1 expression of hair follicular cells (*) in dermis of RASSF92/2 skin. (B) Images at higher magnification, with follicular sites of abnormal K1 expression identified by asterisks (*) in both length-wise sections (top panels) and cross sections (bottom panels) of hair follicles. Scale bar = 40 mm. Blue, DAPI staining of cell nuclei. doi:10.1371/journal.pone.0017867.g008 RASSF9 recombinant virus inhibited the BrdU incorporation in RASSF92/2 keratinocytes (P,0.05; Figure 12B). These results show that RASSF9 is involved in suppressing or regulating proliferation of epidermal keratinocytes. As described above, RASSF9 expression was prominent in suprabasal layers of epidermis, and RASSF9 mice exhibited aberrant differentia- tion. These observations prompted us to test whether RASSF9 was actively involved in keratinocyte differentiation. A high concen- tration of calcium (2 mM) was used to induce the differentiation of primary mouse keratinocytes in culture [10], and two markers, loricrin and filaggrin, were used for evaluation of terminal differentiation. We found that both loricrin and filaggrin were expressed at lower levels in RASSF92/2 versus WT keratinocytes, regardless of calcium concentrations and incubation lengths tested (4 days, Figure 12C; 2 days, Figure S5A). In cells cultured in 2 mM calcium for 4 days, the levels of filaggrin and loricrin in RASSF92/2 keratinocytes were about two-third and one-third, respectively, of those in WT keratinocytes (Figure 12C). These results suggest that the terminal differentiation process of RASSF92/2 keratinocytes is deficient. PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17867