transfected in triplicates with Fugene 6 (Roche) and TOP/FOP or Hes1-luc plasmids with addition of forced expressing active Notch1 (N1IC) or Notch2 (N2IC) pcDNA3 plasmids and pRL- TK (Promega). Luciferase activity was measured with the Dual- luciferase reporter assay system (Promega), with the Renilla luciferase (pRL-TK) activity as an internal control, 48 h after transfection. The experiment was repeated three times, the mean of all results was taken and expressed as a percentage of induction over control ( = 100%). Wounding and preparation of wound tissue Skin wound healing analysis was performed as described previously [58]. Briefly, full-thickness excisional skin wounds (6 mm in diameter) were made in WT mice. Animals were killed 5 days after wounding (n = 4), and an 8–10 mm area, including the complete epithelial margins, was collected and used for histopath- ological analysis. Three small areas (363 mm) of wounded and unaffected skin from the same animal were used to prepare RNA for expression analysis. Four mice were analyzed. Quantitative RT-PCR RNA was isolated from primary keratinocytes using Qiagen RNeasy Isolation Kit followed by cDNA synthesis (SuperScript II, Invitrogen). Real-Time PCR was performed with 800 nM primers diluted in a final volume of 20 ml in SYBR Green Reaction Mix (Applied Biosystems). RT-PCRs were performed as follows: 95uC for 10 min, 45 cycles of 95uC for 10 sec, 60uC for 10 sec and 72uC for 10 sec. using LightCycler 480 (Roche). All samples were analyzed in triplicate. Cyclophilin and HPRT were used for normalization. The following primers were used: K6a-F 59-GAGCTGGCTTTGGTGGTG-39 References 1. Gannon M, Herrera PL, Wright CV (2000) Mosaic Cre-mediated recombina- tion in pancreas using the pdx-1 enhancer/promoter. Genesis 26: 143–144. 2. Gu G, Dubauskaite J, Melton DA (2002) Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129: 2447–2457. 3. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, et al. (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4: 437–450. 4. Gannon M, Gamer LW, Wright CV (2001) Regulatory regions driving developmental and tissue-specific expression of the essential pancreatic gene pdx1. Dev Biol 238: 185–201. 5. Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, et al. (2002) Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem 277: 11225–11232. 6. Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371: 606–609. 7. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, et al. (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122: 983–995. 8. Larsson LI, Madsen OD, Serup P, Jonsson J, Edlund H (1996) Pancreatic- duodenal homeobox 1 -role in gastric endocrine patterning. Mech Dev 60: 175–184. 9. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678–689. 10. Lefort K, Dotto GP (2004) Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression. Semin Cancer Biol 14: 374–386. 11. Dotto GP (2008) Notch tumor suppressor function. Oncogene 27: 5115–5123. 12. Lin MH, Leimeister C, Gessler M, Kopan R (2000) Activation of the Notch pathway in the hair cortex leads to aberrant differentiation of the adjacent hair- shaft layers. Development 127: 2421–2432. 13. Uyttendaele H, Panteleyev AA, de Berker D, Tobin DT, Christiano AM (2004) Activation of Notch1 in the hair follicle leads to cell-fate switch and Mohawk alopecia. Differentiation 72: 396–409. 14. Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, et al. (2003) Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 33: 416–421. 15. Pan Y, Lin MH, Tian X, Cheng HT, Gridley T, et al. (2004) gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev Cell 7: 731–743. Pdx1 Is Expressed in the Skin K6a-R 59-GTCCTCCACTGTGTCCTG-39 K10-F 59-GCCAGAACGCCGAGTACCAACAAC-39 K10-R 59-GTCACCTCCTCAATAATCGTCCTG-39 Loricrin-F 59-TCACTCACCCTTCCTGGTGC-39 Loricrin-R 59-CACCGCCGCCAGAGGTCTTC-39 Hes1-F 59-AAAATTCCTCCTCCCCGGTG-39 Hes1-R 59-TTTGGTTTGTCCGGTGTCG-39 p21-F 59-CACAGCGATATCCAGACATTCAG-39 p21-R 59-CGGAACAGGTCGGACATCA-39 Pdx1-F 59-TGCCACCATGAACAGTGAGG-39 Pdx1-R 59-GGAATGCGCACGGGTC-39 Cyclophillin-F 59-ATGGTCAACCCCACCGTGT-39 Cyclophillin-R 59-TTCTGCTGTCTTTGGAACTTTGTC-39 Hprt-F 59-GACCGGTCCCGTCATGC-39 Hprt-R 59-CATAACCTGGTTCATCATCGCTAA-39 Acknowledgments We thank Reinhard Fa¨ssler, Ramin Massoumi and Rudolf A. Rupec for helpful discussions and expertise help with mouse models as well as keratinocytes isolation and culture. The Notch2 antibody developed by S. Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank. We thank W. Gao, Y. Hamada, C.A. Klug and J. Takeda for providing Notch1-GFP, Notch2LacZ and Keratin5-Cre mice respectively. We are grateful to C.V. Wright for the PDX1 antibody. We thank M. Neuhofer and S. Ruberg for excellent technical assistance. Author Contributions Conceived and designed the experiments: PKM JTS. Performed the experiments: PKM. Analyzed the data: PKM BMG BS RS JTS. Contributed reagents/materials/analysis tools: HN UZS FR. Wrote the paper: PKM JTS. 16. Vauclair S, Nicolas M, Barrandon Y, Radtke F (2005) Notch1 is essential for postnatal hair follicle development and homeostasis. Dev Biol 284: 184–193. 17. Demehri S, Turkoz A, Kopan R (2009) Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment. Cancer Cell 16: 55–66. 18. Xia X, Qian S, Soriano S, Wu Y, Fletcher AM, et al. (2001) Loss of presenilin 1 is associated with enhanced beta-catenin signaling and skin tumorigenesis. Proc Natl Acad Sci U S A 98: 10863–10868. 19. Blanpain C, Lowry WE, Pasolli HA, Fuchs E (2006) Canonical notch signaling functions as a commitment switch in the epidermal lineage. Genes Dev 20: 3022–3035. 20. Moriyama M, Durham AD, Moriyama H, Hasegawa K, Nishikawa S, et al. (2008) Multiple roles of Notch signaling in the regulation of epidermal development. Dev Cell 14: 594–604. 21. Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, et al. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10: 547–558. 22. Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, et al. (2007) Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation. J Exp Med 204: 331–343. 23. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71. 24. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, et al. (2005) Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7: 469–483. 25. Greenhalgh DA, Rothnagel JA, Quintanilla MI, Orengo CC, Gagne TA, et al. (1993) Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene. Mol Carcinog 7: 99–110. 26. Vitale-Cross L, Amornphimoltham P, Fisher G, Molinolo AA, Gutkind JS (2004) Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer Res 64: 8804–8807. 27. Tuveson DA, Shaw AT, Willis NA, Silver DP, Jackson EL, et al. (2004) Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 5: 375–387. 28. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, et al. (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19: 245–254. PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13578