pyranoside (IPTG) for 4 hr at 37uC, and the resulting GST- RASSF9 fusion protein with a GST tag in the N-terminus were purified and used to generate antiserum in rabbits. Specificity of anti-RASSF9 antibody is confirmed by Western immunoblotting of exogenously expressed RASSF9 protein (Figure S1). Histology and immunofluorescence Dorsal skin of mouse was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 mm. The sections were deparaffinized, rehydrated, and then subjected to H&E staining or Masson’s trichrome staining. The resulting images were examined and photographed under a microscope (Axioplan II; Carl Zeiss Microimaging Inc., Germany). For immunofluorescence analysis, OCT-embedding frozen skin was cut into 4-mm sections, fixed in ice-cold acetone for 30 sec (for RASSF9 immunofluorescence staining) or neutral buffered formalin for 15 min, and subjected to double-indirect immuno- staining according to the IHC World protocol (http://www. ihcworld.com/protocols/general_IHC). Finally, the specimens were mounted with Vectashield (Vector Labs, Burlingame, CA) and photographed by laser-scanning confocal microscopy as earlier described. The primary antibodies used in these experi- ments were as follows: anti-keratin 1, keratin 5, keratin 6, keratin 10 and keratin 14 polyclonal antibodies (Covance, Berkeley, CA); rabbit anti-RASSF9 (generation described in Materials and Methods). The utilized secondary antibodies were FITC- or TRITC-conjugated (Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were stained with DAPI. Construction of the recombinant adenovirus The full-length RASSF9 gene was obtained from the RNA of normal ICR mouse brain using RT-PCR. The resulting 1.3-kb fragment was cloned into pCMV-HA (Clontech, Palo Alto, CA) and amplified using specific primers designed to generate an HA- tagged RASSF9 fragment. The amplified fragment was subcloned into the NotI-XhoI sites of pShuttle-CMV and inserted into the adenoviral backbone plasmid, pAdEasy-1, for amplification in bacteria. Recombinant adenoviruses containing the RASSF9 gene were generated according to the protocol for the utilized adenovirus-construction system (http://www.coloncancer.org/ adeasy/protocol.htm). A GFP-encoding recombinant adenovirus was used as the control. Expression of RASSF9 in adenovirally infected keratinocytes was confirmed by immunoblotting with anti-HA antibody. Titration of recombinant adenovirus was performed by a plaque forming unit (PFU) assay in 293 cells following the manufacturer’s instructions (Cell Biolabs). Cell culture and viral infection The isolations and incubations of epidermal keratinocytes were done according to a web-published protocol (http://openwetware. org/wiki/Mouse_keratinocyte_cultures) with minor modifications. Briefly, epidermis was isolated from the skin of neonatal mice by incubation with Dispase II overnight at 4uC. Epidermal keratinocytes were dissociated by incubation of the skin samples in Trypsin-EDTA for 20 min at room temperature, and the cells were harvested by passing each sample through a sterile nylon 100-mm cell strainer (BD Falcon, Bedford, MA). The epidermal keratinocytes were then centrifuged, resuspended, plated onto collagen pre-coated dishes, and cultured in Eagle’s minimal essential medium (EMEM; Cambrex, East Rutherford, NJ) supplemented with 4% Chelex-100 treated fetal bovine serum (FBS) (Bio-Rad Laboratories, Hercules, CA), 0.4 mg/mL hydro- cortisone, 5 mg/mL insulin, 10 ng/mL epidermal growth factor (EGF), 10210 M cholera toxin, 261029 M triiodothyronine (T3), RASSF9 Regulated Epidermal Homeostasis 100 units/mL penicillin, 100 mg/mL streptomycin, 2 mM L-Glu, and 0.06 mM CaCl2. After 17 hr, the culture medium was refreshed, and the cells were exposed to the recombinant adenovirus. The inoculating medium was removed 24 hr post infection. After infection, the cells were incubated in fresh media containing 0.06 mM or 2 mM calcium for the indicated time. Recombinant adenoviruses were produced and routinely used at a multiplicity of infection (MOI) of 10, unless otherwise specified. Western blotting Adherent primary cells were harvested and homogenized at 4uC for 20 min in RIPA lysis buffer (25 mM TRIS, pH 7.5, 150 mM NaCl, 1% NP40, 1% Na-deoxycholate, and 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.3 mM aprotinin, 130 mM bestatin, 1 mM EDTA, and 1 mM leupeptin (Sigma Chemical Corp., St Louis, MO). Each lysate was cleared of cell debris by centrifugation, and each sample’s total protein concentration was determined using the Bradford protein assay reagent (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved by electrophoresis on SDS-polyacrylamide gels (Bio-Rad, Hercules, CA), and then transferred to a polyvinylidene difluoride membrane (PVDF) (Millipore Corp., Bedford, MA). Immunoblot- ting was performed as previously described [14] using the following specific primary antibodies: anti-filaggrin, anti-loricrin, anti-keratin 14 (Covance, Berkeley, CA); anti-HA (Sigma Chemical Corp., St Louis, MO) and anti-p21Cip1 (F-5; Santa Cruz, CA). The proteins of interest were reacted with a horseradish peroxidase-conjugated secondary antibody, and then detected with an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). K14, which is constitutively expressed in keratinocytes irrespective of the cell-growth condi- tions, was used as the loading control. In situ hybridization Fresh dorsal skin tissue was embedded in optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA), and 4-mm cryosections were cut and subjected to in situ hybridization according to the protocols of the Anderson lab (California Institute of Technology, Pasadena, CA; http://wmc. rodentia.com/docs/Big_In_Situ.html). Fragments corresponding to nucleotides (nt) 285–817 and 1126–1625 of the mouse RASSF9 cDNA (NCBI RefSeq NM_146240) were separately cloned into the EcoRI-XhoI sites of the pBluescript II KS (+) phagemid vector (Stratagene, La Jolla, CA) for riboprobe synthesis. Digoxigenin- UTP (DIG)-labeled antisense and sense riboprobes were prepared by in vitro transcription with DIG-11-UTP and either T3 or T7 RNA polymerase, according to the manufacturer’s protocol (DIG Application Manual for In Situ Hybridization, 3rd edition; Roche Molecular Biochemicals, Mannheim, Germany). The hybridiza- tion signal was detected as a dark precipitate that formed following exposure to an alkaline phosphatase-labeled anti-DIG antibody and a substrate containing nitroblue tetrazolium (NBT) (Promega, Madison, WI) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Promega, Madison, WI). Affymetrix Analysis Total RNA was extracted from mouse primary keratinocytes of the neonatal WT (n = 1) and RASSF92/2 mice (n = 2) by TRIzol extraction per manufacturer’s instructions(Invitrogen). Briefly, one 10-cm culture plate of confluent monolayer of primary keratino- cytes per sample was lysed in 1 mL TRIzol reagent after maintenance for two days in low-calcium keratinocyte serum-free medium with supplements (Invitrogen, Carlsbad, CA). The cell lysate was mixed with 0.2 mL of chloroform and centrifuged at PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17867