RASSF9 Regulated Epidermal Homeostasis Figure 7. Aberrant differentiation in the skin of RASSF92/2 mice—K14 (red). (A) Frozen sections of dorsal skins of WT (+/+) and RASSF92/2 (2/2) mice were immunostained with red fluorescence for K14 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. (B) Images at higher magnification. Scale bar = 40 mm. Blue, DAPI staining of cell nuclei. doi:10.1371/journal.pone.0017867.g007 profiles of RASSF9 gene in various organs of WT mice at one or two weeks old. Our results indicated that the RASSF9 mRNA was expressed in multiple organs, with high-level expression seen in the skin, moderate-to-high expression in the heart, lung and kidney, and relatively low expression in the thymus, brain, stomach, liver, intestine and spleen (Figure 11A). Interestingly, RASSF9 mRNA expression in heart and lung increased with growth from one to two weeks old, implicating a role of this gene in regulation of post- partum maturation of these organs in addition to epidermis development. We then isolated epidermal and dermal fractions from the skin of 4-dpp WT mice and examined the site-specific expression of RASSF9 mRNA in skin. The RASSF9 mRNA was between two- and six-fold higher in the epidermal fraction than the dermal fraction, depending on the reference gene used (Figure 11B, top panel). The demarcation of epidermis and dermis layers was confirmed by specific detection of mRNA for E- cadherin (CDH1) and fibronectin (fn1) in the former and latter fractions, respectively (Figure 11B, middle and bottom panels). We further visualized the expression patterns of RASSF9 protein in mouse skin tissues by double immunofluorescence staining of frozen sections using RASSF9- and K1-specific antibodies. Western immunoblot detection of exogenously ex- pressed RASSF9 confirmed the specificity of anti-RASSF9 antiserum (Figure S1). The RASSF9-specific signal was detected throughout the entire epidermis of wild type (WT, +/+) mice at 4- dpp while colocalizing with K1 in the suprabasal layer (Figure 11C; higher-magnification images in Figure 11D). Immunofluorescence signals specific for RASSF9 were not detected in the RASSF92/2 skin section, again demonstrating the specificity of the anti- RASSF9 antiserum [Figure 11D; compare +/+ (left panels) with 2/2 (right panels)]. Quantification of the RASSF9 immunoflu- orescent intensity using ImageJ software revealed a more prominent expression of RASSF9 in suprabasal layers, compared to that in the basal and granular layers (Figure 11C). In situ hybridization with an antisense probe against the RASSF9 mRNA further confirmed strong and specific expression of RASSF9 that is prominent in suprabasal layer of normal mouse epidermis (Figure S4). RASSF9 affects keratinocyte growth and differentiation in vitro Since RASSF9 is expressed in the proliferating basal and differentiating suprabasal epidermal layers, it may have significant involvement in keratinocyte proliferation and differentiation. Therefore, we isolated primary keratinocytes from RASSF92/2 mice and measured their BrdU incorporation. A moderate (1.5- fold) but statistically significant increase in BrdU incorporation (p,0.05) was detected in RASSF92/2 keratinocytes versus WT keratinocytes, in terms of both extent of BrdU incorporation and the percentage of BrdU-positive cells (Figure 12A). Converse- ly, re-expression of RASSF9 by infection with the Adv/HA- PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17867