expression in RASSF92/2 mice means that p21Cip1 expression is not sufficiently up-regulated in keratinocytes progressing to early differentiation, allowing these keratinocytes to escape from cell-cycle withdrawal and potentially accounting for the epidermal hyperpla- sia observed in these mice (Figure 14B). Supporting Information Figure S1 Specificity of rabbit anti-RASSF9 antiserum. RASSF92/2 primary keratinocytes were transduced with Adv/ GFP (GFP) or Adv/HA-RASSF9 (HA-RASSF9) at indicated MOI and incubated in low (2, 0.06 mM) or high (+, 2 mM) calcium ion (Ca2+) concentration. Protein lysates of transduced cells were subjected to SDS-PAGE and Western immunoblot using the RASSF9-specific rabbit antiserum generated as described in Materials and Methods at a dilution of 1:1000. The membrane was reprobed for Keratin-14 (K14) as loading control. In both calcium conditions a major band near 60 kDa, the approximate size of HA-RASSF9, was detected by anti-RASSF9 antiserum only for cells transduced with Adv/HA-RASSF9. The molecular weights of the protein size markers labeled to the right of the blot are, starting at the top, 170, 130, 100, 70, 55, 40, 35, 25, 15 kDa. (TIF) Figure S2 LMP-1 and RASSF9 gene expressions in LMP-1 transgenic mice. (A) Skin tissue RNA samples of heterozygous and homozygous transgenic mice of 1 day post-partum (dpp; n = 5) and 4 dpp (n = 3) were subjected to RT-PCR for amplification of LMP-1 and RASSF9 transcripts. Separately, mouse genotyping was done by PCR of tail DNA using the specific primers for the detection of LMP- 1 transgene insertion and RASSF9 intron deletion. In the cDNA samples of both heterozygotes (1-dpp: Tg-1 and Tg-2; 4-dpp: Tg-6 and Tg-7) and homozygotes (1-dpp: Tg-3 – Tg-5; 4-dpp: Tg-8) no LMP-1 transcript was detected. RASSF9 transcript and the deleted intron sequence targeted by the transgene insertion had been lost in the homozygote samples. For the cDNA samples, NIH-3T3 cells overexpressing full-length LMP-1 and skin tissue of WT mice at 7 dpp were used as positive controls of LMP-1 and RASSF9 transcriptions, respectively. Tail genomic DNA extracts of genotyp- ing-confirmed heterozygous mice were used as positive control of presence of both LMP-1 transgene insertion (‘‘LMP-1’’) and transgene-replaced intron deletion of RASSF9 (‘‘del’’). b-Actin was used as internal control. (B) Protein expressions of LMP-1 in LMP-1 transgenic mice at two weeks old were examined by immunoblot with anti-LMP-1 antibody. In both WT mice and the two transgenic mouse lines, Tg1 and Tg2, no LMP-1 protein expression was detected. The asterisk (*) denotes the founder mouse line subsequently used for the experiments reported in this study. 293T cells transfected with LMP-1 expression plasmid constructs pT7(E) and D94 [46,47], and B95-8, the EBV-infected marmoset B-lymphoblastoid cell line [48], were used as positive controls of LMP-1 protein expression. The blot was reprobed for b-Actin as protein loading controls. (TIF) Figure S3 Histological abnormalities in RASSF92/2 skin. Masson’s trichrome staining of skin sections from two-week- old mice. The dashed yellow lines denote the epidermis-dermis and dermis-adipose borders. Similar results were obtained from three independent pairs of mice. The black dashed line-framed areas are enlarged in the panels on the right. Scale bar = 100 mm. Epi, epidermis; Der, dermis; Adi, adipose. (TIF) Figure S4 In situ hybridization of RASSF9 mRNA in the dorsal skin of mice. Skin cryosection of two-week-old RASSF9 Regulated Epidermal Homeostasis mice were fixed in paraformaldehyde for in situ hybridization assay. Top right panel: a positive brown signal was developed using an antisense probe against the mRNA for RASSF9 in WT mice. Top left panel: a sense probe was used as a negative control; no signal was detected in WT mice. Bottom panel: no signal was detected in RASSF92/2 mice, irrespective using a sense probe (left) or antisense probe (right), which further confirmed the probe specificity in this system. Similar results were obtained in two independent experiments. Arrow, positive signal in WT epidermis. Scale bar = 100 mm; +/+, wild type; 2/2, RASSF92/2. (TIF) Figure S5 RASSF9 mediates the terminal differentiation of keratinocytes. (A) The terminal differentiation of RASSF92/2 (2/2) versus WT (+/+) mouse keratinocytes was analyzed by immunoblotting with specific antibodies against filaggrin and loricrin. K14 was used as the loading control for keratinocytes. Total cell extracts were prepared from mouse primary keratinocytes incubated for 2 days under growth (0.06 mM calcium) or differentiation-inducing (2 mM calcium) conditions. The intensity of protein expression was determined as the density of the relevant band normalized with respect to that of the K14 loading control; the analysis was performed using the ImageQuant 5.1 software, and the results are shown below the panel. (B) Re-expression of RASSF9 in primary RASSF92/2 keratinocytes. Primary RASSF92/2 kerati- nocytes were infected with Adv/HA-RASSF9 or a GFP-expressing control virus for 24 hr in low-calcium medium, and then switched to fresh growth (0.06 mM calcium) or differentiation-inducing (2 mM calcium) medium for 2 days. The expression levels of the terminal differentiation markers were detected as described in (A). Similar results were obtained in three independent experiments. +/+, wild type; 2/2, RASSF92/2. (TIF) Figure S6 RASSF92/2 mice with a moderate impair- ment of skin barrier. Skin permeability assays on RASSF92/2 and control newborn pups were performed by toluidine blue dye- penetration assays. RASSF92/2 pups showed moderate impaired skin barrier in RASSF9 deficient pups (2/2), which exhibited slight increase in dye penetration with a punctuated distribution as compared with that of the WT control (+/+). Bottom panel was the enlarged image of RASSF92/2 mice marked by * shown in the top panel. Red arrows, signals of dye-penetration; +/+, wild type; +/2, RASSF9+/2; 2/2; RASSF9 2/2. (TIF) Figure S7 Overexpression of LMP-1 could not reduce the filaggrin expression. (A) Normal mouse primary kerati- nocytes were infected with adenoviral vectors encoding LMP-1 or GFP at MOI’s = 5 or 10 in low-calcium concentration (0.06 mM) for one-day infection, followed by incubation for 4-day in high- calcium medium (2 mM) as previously described prior to lysis and Western immunoblotting. (B) Intensity measurements of the filaggrin bands. Data represent two separate blots. No statistical differences in band intensities were detected. (TIF) Figure S8 Histological abnormalities in developing RASSF92/2 lung. H&E staining of lung sections of mice at two weeks old. The RASSF92/2 lung (2/2) exhibits stunted progress of saccule septation versus WT lung (+/+), with grossly enlarged alveoli. In the panels of RASSF92/2 section noticeable thickening of pulmonary alveolar epithelium can be observed (asterisks, *). (A) Low-magnification views of pulmonary alveoli. PLoS ONE | www.plosone.org 19 March 2011 | Volume 6 | Issue 3 | e17867