The results of our in vitro study of mouse primary keratinocytes showed that RASSF9 mediated growth suppression and activation of the differentiation program. The mechanism by which RASSF9 mediates keratinocyte growth suppression may rely on the regulation of cell-cycle inhibitor p21Cip1, as demonstrated by the results of reciprocal alterations between deficient RASSF9 expression and its compensation in mouse primary keratinocytes. Taken together, our findings show that RASSF9 is essential for the maintenance of epidermal homeostasis. Materials and Methods Animals All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Chang Gung University, Taiwan (Permit Number: CGU10-027), and were carried out in accordance with the relevant guidelines. The mice were bred and maintained under a consistent temperature of 21– 23uC, a relative humidity of 50–70%, and a 12-hr light-dark cycle with normal access to water and chow. In all experiments, mice were sacrificed by ether inhalation. Generation of transgenic mice The utilized transgene was the nasopharyngeal carcinoma (NPC)-derived LMP1 (NLMP1) gene, including the upstream promoter, coding sequences, and the 59-untranslated region [13]. Transgenic mice were generated as follows. Briefly, the relevant EcoRI-HindIII fragment was injected into fertilized oocytes from ICR strain mice. Founders were crossed with ICR mice, and PCR and Southern blot analysis were used to screen progeny for the presence of the transgene. Southern blotting For Southern blotting, 20 mg of tail-tissue-derived genomic DNA was digested with either EcoRI or HindIII, which do not cut the transgene. The digests were resolved by electrophoresis, and the DNA was transferred to a nylon membrane (Hybond-N, Amersham Biosciences, Piscataway, NJ). Probes were prepared using the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals, Mannheim, Germany). The probes were designed from the transgene sequence or the mutated genome, and were designated Tg_probe or D_probe, respectively (Table S1). Southern blotting was performed according the DIG system’s protocol for filter hybridization (Roche Molecular Biochemicals, Mannheim, Germany). Inverse PCR amplification of the transgene-flanking genomic sequences Tail-derived genomic DNA was completely digested with TaqI or BclI, and then self-circularized with T4 DNA ligase (2,5 mg/ mL). The ligation mixture was extracted with phenol:chloroform (1:1,v/v) followed by chloroform alone, and DNA was precipitated with ethanol and sodium acetate (pH 5.2). The self-ligated DNA (1 mg) was used as the template for PCR amplification of the EcoRI and HindIII flanking genome sequence of the insertion fragment. The utilized primers were specific for both end sequences of the transgenic DNA (Supplementary Information Table S1). RNA isolation RNA from tissues and keratinocytes was prepared by TRIzol extraction according to manufacturer’s protocol (Invitrogen, Karlsruhe, Germany). RASSF9 Regulated Epidermal Homeostasis RT-PCR and QRT-PCR One mg of total RNA was subjected to reverse transcription using an oligo-dT primer (Invitrogen, Carlsbad, CA). The reaction mixture was then amplified using gene-specific intron-crossing oligonucleotide primer pairs specific to the genes encoding mouse RASSF9, p21Cip1 and the reference genes of b-Actin, 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltrans- ferase (HPRT1). Quantitative RT-PCR was performed on a LightCycler (Roche Molecular Biochemicals, Mannheim, Ger- many) according to manufacturer’s instructions, using the FastStart DNA Master SYBR Green I mix (Roche Molecular Biochemicals, Mannheim, Germany). The amplification condi- tions were as follows: 95uC for 10 minutes followed by 45 cycles of 95uC for 10 seconds, 60uC for 10 seconds, and 72uC for 10 seconds. The cycle threshold (Ct) values were normalized with respect to those of the reference genes. The oligonucleotide sequences of the utilized primers are listed in Supplementary Information Table S1. BrdU-incorporation assays in vivo and in vitro Mice were in vivo pulse-labeled with BrdU (250 mg/g body weight; Sigma Chemical Corp., St Louis, MO) via intraperitoneal (i.p.) injection, and skin tissues were harvested after two-hr incubation. Each sample was fixed in formalin, embedded in paraffin, and sectioned at 4 mm. The sections were deparaffinized, rehydrated, subjected to antigen retrieval in citrate buffer, and then subjected to immunohistochemical staining using specific antibodies against BrdU (Serotec, Oxford, UK) and keratin 14 (Covance, Berkeley, CA) according to the protocol for the utilized anti-bromodeoxyuridine-fluorescein kit (Roche Molecular Bio- chemicals, Mannheim, Germany), with the DNA-denaturation step modified to incubating the slides in 2 M HCl, for 20 min at 37uC. Chicken anti-rat IgG conjugated with FITC was used for BrdU detection (Santa Cruz, CA), while goat anti-rabbit IgG conjugated with TRITC was used for K14 detection (Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were stained with 496-diamidin-2-phenylindol-dihydrochloride (DAPI; Sigma Chemical Corp., St Louis, MO). The resulting images were examined and photographed under a laser-scanning confocal microscope (Leica TCS SP2; Leica, Germany or LSM 510 Meta NLO; Carl Zeiss, Inc., USA). For the in vitro assay, mouse primary keratinocytes were seeded to 48-well plates and incubated with growth medium (0.06 mM calcium) for 17 hr, followed by infection with or without a RASSF9-encoding recombinant adenovirus for 24 hr. Subse- quently, the cultures were plated with fresh medium containing 0.06 mM calcium and incubated for another 48 hr. Keratinocytes were pulse-labeled with 10 mM BrdU for 2 hr and then harvested, and BrdU incorporation was analyzed by ELISA according to the manufacturer’s protocol (Cell Proliferation Biotrak ELISA System; Amersham Biosciences, Piscataway, NJ). The BrdU immunoflu- orescence staining was performed according to the protocol described above. Bacterial expression of RASSF9, and anti-RASSF9 antibody preparation The 285-1625nt fragment of the mouse RASSF9 cDNA (NCBI RefSeq NM_146240) was cloned into the EcoRI-XhoI sites of the pGEX-4T-1 vector (Amersham Biosciences, Piscataway, NJ). The resulting pGEX-4T-1-RASSF9 plasmids were transformed into Escherichia coli BL21 competent cells and grown to the appropriate cell density at 37uC with Ampicillin selection. Protein expression was induced by addition of 1 mM isopropyl-1-thio-b-D-galacto- PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17867