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DMBA/TPA Treatment Is Necessary for BCC Formation from Patched Deficient Epidermal Cells in Ptchflox/floxCD4Cre+/− Mice  Anja Uhmann, Ina Heß, Anke Frommhold,

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Presentation on theme: "DMBA/TPA Treatment Is Necessary for BCC Formation from Patched Deficient Epidermal Cells in Ptchflox/floxCD4Cre+/− Mice  Anja Uhmann, Ina Heß, Anke Frommhold,"— Presentation transcript:

1 DMBA/TPA Treatment Is Necessary for BCC Formation from Patched Deficient Epidermal Cells in Ptchflox/floxCD4Cre+/− Mice  Anja Uhmann, Ina Heß, Anke Frommhold, Simone König, Sebastian Zabel, Frauke Nitzki, Kai Dittmann, Fred Lühder, Hans Christiansen, Julia Reifenberger, Walter Schulz-Schaeffer, Heidi Hahn  Journal of Investigative Dermatology  Volume 134, Issue 10, Pages (October 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Histological analyses of DMBA/TPA-induced BCC. Anti-CD3 immunhistological stainings of (a) DMBA/TPA-induced BCC of Ptchflox/floxCD4Cre+/− and (b) untreated control skin. Arrows: dermal CD3+ cells; dashed arrows: epidermal CD3+ cells in the basal layer; inset: tumor-infiltrating CD3+ cells. (c–f) In situ hybridizations to visualize the expression of the Ptch (477bp probe in c), the wt Ptch (250bp probe in d and e), or Gli1 (f) transcripts of DMBA/TPA-induced BCC of Ptchflox/flox CD4Cre+/− skin. Panel d shows wt Ptch expression in the hair follicle (arrows). For detailed explanation of the 477-bp and 250-bp Ptch probes, see the main text. Note that a, c, and e are adjacent sections of the same BCC. Bar = 200μm. BCC, basal cell carcinoma; DMBA, 7,12-dimethylbenz(a)anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 RFLP analyses of the Hras G61L mutation and pERK1/2 immunohistological staining of DMBA/TPA-induced BCC and papilloma. (a) Amplification of Hras exon 2 (upper lane), and subsequent RFLP analyses by AvaII or XbaI digestion (see main text, Supplementary Figure S5 and the Supplementary Methods section online) of genomic DNA isolated from papilloma of DMBA/TPA-treated Ptchflox/flox mice (pap1-5) and from papilloma and BCC of DMBA/TPA-treated Ptchflox/floxCD4Cre+/− mice (pap1-5 and BCC1-5, respectively). Genomic DNA from a wild-type mouse (wt) or a BCC of a Ptchflox/floxCreERT2+/−mouse (BCC) (Zibat et al., 2009) served as controls. Asterisks: Hras G61L-positive papilloma. (b–d) Anti-pERK1/2 immunohistological stainings of papilloma (b and c) and BCC (d) of DMBA/TPA-treated Ptchflox/floxCD4Cre+/− mice. Arrows: pERK1/2-positive papilloma; asterisk: BCC. Bar = 200μm. BCC, basal cell carcinoma; DMBA, 7,12-dimethylbenz(a)anthracene; RFLP, restriction fragment length polymorphism; TPA, 12-O-tetradecanoylphorbol-13-acetate. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 LacZ staining and Gli1 in situ hybridization for the Gli1 expression of DMBA/TPA-induced BCC from Ptchflox/floxCD4Cre+/−R26RT/+ mice. (a) LacZ-stained DMBA/TPA-induced BCC of Ptchflox/floxCD4Cre+/−R26RT/+ skin. Asterisk: LacZ-negative BCC. (b) Gli1 expression of LacZ+ BCC and (c) of LacZ- BCC. Bar = 200μm. BCC, basal cell carcinoma; DMBA, 7,12-dimethylbenz(a)anthracene; LacZ, β-galactosidase; TPA, 12-O-tetradecanoylphorbol-13-acetate. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Flow cytometric analysis of keratinocytes isolated from the back skin of Ptchflox/flox mice. Keratinocytes of the back skin of Ptchflox/flox mice were isolated and stained with anti-CD4, anti-CD49f, anti-CD34, and anti-Sca1 antibodies, as described in the Materials and Methods section. (a) Cells were divided into a CD4-negative and a CD-positive fraction. (b) On the basis of SSC/FSC, keratinocytes and lymphocyte fractions were distinguished and were separately divided into CD49fhigh (I) and CD49flow (II) fractions. Subsequently, these fractions were analyzed for CD34 and Sca1 expression (Jensen et al., 2010) (for analysis of the lymphocyte fraction see Supplementary Figure S11 online). The overlays of the CD34/Sca1 plots of the CD4+ and the CD4- fractions revealed a CD4+ CD49fhigh Sca+ cell population (0.23% of all keratinocytes) that expresses intermediate levels of CD34. FSC, forward scatter cytometry; SSC, side scatter cytometry. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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