RASSF9 Regulated Epidermal Homeostasis Figure 5. K6 is abnormally expressed in epidermis of RASSF92/2 skin. (A) Frozen sections of skin from two-week-old mice were double- immunofluorescence stained for K6 (red) and K14 (green), with the merged signals in yellow. DAPI (blue) was used to for nuclear staining. Scale bar = 100 mm. Similar results were obtained from three independent mice. +/+, wild type; 2/2, RASSF92/2. The other abbreviations of Epi, Derm, Adi were defined as described in Figure 3A. Areas outlined in white borders were enlarged for detailed views (insets). (B) High-magnification images of K6 and K14 expression, prepared as described above. Scale bar = 20 mm. doi:10.1371/journal.pone.0017867.g005 mice. BrdU, the thymidine analog, incorporates into replicating DNA; it is useful as a label of proliferating cells. Indeed, immunofluorescence staining for BrdU-incorporated cells in skin tissues revealed marked changes of cellular proliferation patterns in the basal layers and hair follicles of RASSF92/2 mice versus WT. (Figure 4A and B). BrdU incorporation doubled in the epidermal basal layer of the RASSF92/2 mice versus WT control (Figure 4A and B; 4C, top panel). Additionally, BrdU- positive cells were 4-fold more in the outer root sheath (ORS) of the hair follicles located at the region of dermis layer in RASSF92/2 mice (Figure 4A and B; 4C, middle panel). However, the BrdU-labeling hair bulb index, defined by the ratio of the number of hair bulb (HB) with more than five BrdU- immunolabeled nuclei to total number of HB, was reduced to ,15% of the WT control (Figure 4A and B; 4C, bottom panel), revealing a remarkable loss of anagenic follicles in RASSF92/2 PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17867