s. Preparation of mouse embryonic fibroblast and keratinocytes On day 13.5 of gestation, embryos from JWA/D26JWA/D2 crossed females were harvested. Embryo head and all visible organs were removed. Remaining embryo was put in 
50 ml tube and minced with scissors. 5 ml 0.25% trypsin was added and incubated in 37uC water bath for 20 min. Trypsin was inactivated using 5 ml of Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. The harvested cells were centrifuged to pellet at 15006g for 5 min and then resuspended with 15 ml of fresh medium. Cell suspension was allowed to stand for 510 min to let the debris settle to the bottom. Top 10 ml of medium containing cells was removed and plated in a 100-mm dish. For isolating keratinocytes, full-layer skin removed from buy ATL 962 newborn mice was treated with 0.25% trypsin Western blot analysis Papillomas and dorsal skin of the mice were isolated and homogenized with a grinder for 10 min in RIPA lysis buffer as JWA Is Required for Induction of Skin Papillomas previously described. Cellular protein was extracted by whole cell extract protocols from cell pellets in protein lysis buffer containing protease and phosphatase inhibitors. Western blot analysis was performed by standard procedures. Membranes were incubated with antibodies detecting phosphorylated B-Raf, MEK, ERK1/2, JNK, p38, total B-Raf, MEK, ERK1/2, JNK, p38 and a-tubulin; JWA, Elk1, c-fos, c-myc; PCNA, bactin tumor numbers. In all the above analyses, a P value of,0.05 was considered statistically significant. Results Targeted disruption of the mouse JWA gene To investigate the role of JWA in the development of mammalian skin tumors, we constructed 12624814” the conditional JWA knockout mice. Exon2 of JWA was floxed with Loxp site, after Cre mediated recombination, the exon2 was deleted . The conditional JWA knockout mice were produced by intercrossing the JWAD2/ mice. Genotyping of Loxp was shown in Fig. 1B and Fig. 1C and genotyping of EIIa Cre was indicated in Fig. 1DE. Genotyping of JWA knockout mice were identified at genome DNA level; mRNA level and Statistical analysis Data were analyzed by Prism software 5.0. The Kaplan-Meier method was used for comparison of the tumor development induced by DMBA/TPA. The Student’s t-test was performed to determine statistical significance for neutral comet assay and relative expression levels. Wilcoxon rank-sum test was used to compare the difference in 4 JWA Is Required for Induction of Skin Papillomas protein level. Although the JWAD2/D2 mice developed premature ageing like phenotypes such as decreased body weight, kyphosis, osteoporosis, and immune organ atrophy, we have not found any spontaneous tumors during their lifespan. Fig. 2 C and D). Skin specimens with H&E staining confirmed that the papillomas with no significant difference between the mice. These data suggest that JWA deficiency attenuates the initiation and development of mouse skin papillomas induced by DMBA/TPA treatment. JWA deficiency attenuates the development of mouse skin papillomas As shown in Fig. 2A, skin papilloma was induced by DMBA/ TPA treatment in both JWA/ and JWAD2/D2 mouse skin. In the present 15363972” study, the first papilloma was observed after 8 weeks of TPA treatment in JWA/ mouse and appeared 2 weeks later in JWAD2/D2 mouse than in JWA/ mouse. At the 19th week of TPA treatment, the end point of experiment, 11 JWA/ mice and 6 JWAD2/D2 mice developed skin papillomas. The ratio of tumor induction in JWA/ mice was significant
