ents. All experiments were conducted in accordance with Animal Care and Use Committee of Model Animal Research Centre. For mice and cells genotyping, genomic DNA from the tip of tail of 4-week-old pups or cells was extracted by means of standard protocols.JWA Is Required for Induction of Skin Papillomas For mouse and cells genotype confirmation at RNA level, the RNA from mice or cells was transcribed and subjected to RTPCR analysis by standard protocol. The following primers were used for detection of JWA+/+ and JWAD2/D2 mice: 59-AACCGTGTAGTGAGCAATCTTG and YM-155 59-GGATAATGCCCATCGGAGT. The PCR products from genomic DNA and cDNA were subject to further sequencing analysis for final verification. overnight at 4uC. The epidermis was then peeled off from the dermis and minced into pieces smaller than 1 mm, and placed into a sterile flask, dispersed by stirring into single cells for 3060 min, suspended in keratinocyte-SFM with supplements. Cells were first incubated in dishes coated with type I collagen at 34uC in 5% CO2 for 12 h to allow cells to attach. Unattached cells were removed by washing with PBS. Attached cells were further cultured in fresh medium, which was refreshed every 2 days. Skin papilloma induction by DMBA/TPA All the mice used for experiments were maintained in the C57BL/6 background with at least six backcrosses from the original 129Sv/C57BL/6 founder mice. Both wild type and JWAD2/D2 mice were used in this DMBA/TPA two-stage papilloma induction assay. All the genotypes of mice and cells were verified at genomic DNA and cDNA level, respectively. A total of 48 mice were divided into 2 groups, each with either the JWA+/+ or JWAD2/D2 genotype and identical number of male and female mice. To induce skin papillomas, mice were shaved on their dorsal skin, and 2 days later treated topically with 25 mg of DMBA in 100 ml acetone once. One week later, each animal received subsequent topical treatments of 2.5 mg of TPA in 100 ml acetone twice a week for 19 weeks. Treated mice were examined twice a week for detecting the presence of ” skin papillomas, which were not scored as positive until they reached at least 1 mm in diameter. At the end of the two-stage model, all mice were 10554878” sacrificed, and skin papillomas were counted and isolated for further histological analysis. All experiments were conducted in accordance with Animal Care Committee of Nanjing Medical University. Neutral comet assay The keratinocytes were cultured in standard medium for 4 days, and then treated with or without 0.15 mg/ml DMBA for another 24 h. The comet assay was carried out according to the manufacturer’s instructions. Briefly, cells at a concentration of 56105/ml in PBS were mixed gently with pre-melted lowtemperature-melting agarose at a volume ratio of 1 to 9 and spread on glass slides which were coated with normal-temperature agarose. The slides were then submerged in pre-cooled neutral lysis buffer at 4uC for 90 min. After rinsing, the slides were electrophoresed at constant 25 V, 300 mA 
for 20 min, then equilibrated in Tris-borate EDTA solution, and stained with ethidium bromide. Fluorescent images for at least 50 nuclei were captured using an Olympus microscope and analyzed by CASP1.2.2 software for tail moment. Immunofluorescence Cells grown on coverslips for 24 h were treated with or without 0.15 mg/ml DMBA for 24 h. After washing with PBS three times, cells were fixed with methanol for 10 min followed with PBS wash twice, and then incubated in PBS containin
