RASSF9 Regulated Epidermal Homeostasis Figure 9. Aberrant differentiation in the skin of RASSF92/2 mice—K10 (red). (A) Frozen sections of dorsal skins of WT (+/+) and RASSF92/2 (2/2) mice were immunostained with red fluorescence for K10 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 K10 expression of hair follicular epithelium (*) in dermis of RASSF92/2 skin. (B) Images at higher magnification, with follicular sites of abnormal K10 expression identified by asterisks (*) in both length-wise sections (top panels) and cross sections (bottom panels) of hair follicles. Both K1 and K10 are markers of early-stage keratinocyte differentiation, suggesting a delay in epidermal and follicular keratinocyte maturation. Scale bar = 40 mm. Blue, DAPI staining of cell nuclei. doi:10.1371/journal.pone.0017867.g009 To confirm that the defects in the induction of terminal differentiation in RASSF92/2 keratinocytes were due to the loss of RASSF9, we rescued RASSF9 expression by transducing cells with Adv/HA-RASSF9, and examined the differentiation of transduced cells. Our results revealed that filaggrin and loricrin were induced by approximately 8- and 2.5-fold in cells that re- expressed RASSF9 versus those expressing GFP (control), when the cells were cultured under the differentiation-inducing condi- tions (2 mM calcium) for 3 or 4 days (Figure 12D). Notably, expression induction of filaggrin and loricrin by ectopic RASSF9 expression in RASSF92/2 keratinocytes was also observed in growing condition (0.06 mM calcium) for 2 days (Figure S5B), suggesting that RASSF9 expression intrinsically primed cells for initial differentiation independent of calcium levels. Taken together, these findings suggest that RASSF9 is required for proper initiation of keratinocytes differentiation. PLoS ONE | www.plosone.org 12 March 2011 | Volume 6 | Issue 3 | e17867