Health). The rabbit polyclonal antibody against keratin 17 (K17) was a gift of Dr. Pierre Coloumbe (Johns Hopkins University School of Medicine). Western blotting Whole tissue lysates were prepared using Laemmli sample buffer (BioRad Laboratories) or RIPA buffer from BT and control mouse dorsal skins. Equal amounts of lysates were loaded on 10% SDS-polyacrylamide gels and transferred to Immun-Blot PVDF membrane (BioRad Laboratories). After blocking, the membranes were incubated in the specific primary antibodies: anti-HA (1:2000), anti-Ets1 (1:1000), anti-GAPDH (1:10000) or anti- MMP13 (1:200). Antibodies against keratinocyte markers – keratin10, keratin14, involucrin and loricrin -were used at a dilution of 1:5000. After washing, the membranes were incubated with specific horseradish peroxidase (HRP) conjugated secondary antibodies at 1:20,000 dilution. Specific bands were detected by chemiluminescence (KPL or Pierce). Immunohistochemistry and Immunofluorescence For histological analyses, skin samples from various regions of the body were fixed in 4% paraformaldehyde and embedded in paraffin. 4-mm thick sections were then stained with hematoxylin and eosin or Masson’s trichrome reagent. For immunohistochem- istry, 4-mm thick skin sections were deparaffinized and subjected to antigen retrieval by heating. The sections were then immuno- stained with specific primary antibodies - anti-HA (1:300), anti- DNp63 (1:300), anti-Ki67 (1:200), anti-Ets1 (1:150), anti-MMP13 (1:40), anti-keratin 1 (1:500), anti-keratin 5 (1:500), anti-keratin 6 (1:500), anti-involucrin (1:500), and anti-loricrin (1:500) - using the Vectastain ABC kit (Vector labs). Diaminobenzidine (DAB) or Vector Blue (Vector Labs) was used as the enzyme substrate and counter-stained with hematoxylin or Nuclear Fast Red (NFR). For immunofluorescence, 5-mm thick OCT embedded fresh frozen sections were fixed in ice-cold methanol. After blocking, the sections were incubated with anti-keratin6, anti-keratin10, anti- keratin14, anti-loricrin, anti-b4 Integrin or anti-PECAM1 antibod- ies at 1:500 dilution or with anti-Laminin or anti-PCNA antibodies at 1:100 dilution. The sections were then washed and incubated with Alexa-Fluor 568 coupled anti-rabbit IgG (1:750), Alexa-Fluor 488 coupled anti-rat IgG (1:500) or FITC coupled anti-mouse IgG (1:500) antibodies and counterstained with DAPI (49,6-diamidino-2- phenylindole). The immunostained sections were mounted in 80% glycerol in PBS, viewed and photographed at room temperature with an Axiophot Zeiss microscope, equipped with a Hamamatsu ORCA-ER camera linked to SPOT software for image capture. The contrast and brightness of the images were adjusted using the Adobe Photoshop and ImageJ software. Quantitative PCR Analysis Total RNAs were isolated using Trizol (Invitrogen) and treated with DNAse I Turbo (Ambion) to remove genomic DNA contamination. Equivalent quantities of RNA were reverse transcribed using iScript kit (BioRad) and real-time PCR was performed in the ICycler system (BioRad), using iQ SYBR green supermix (BioRad) and gene specific primers. Differences between BT samples and controls were normalized to expression of the ubiquitously-expressed gene b2-microglobulin and determined using the 22DCt method. Reporter gene assays For transient transfections, mouse MK1 keratinocyte cells were plated at a density of 1.26105 cells/well in 6 well plates and were Ets1 in Squamous Neoplasia transfected using Optifect (Invitrogen) or Fugene 6 reagent (Roche Diagnostics) at a density of 40% according to the manufacturer’s instructions. The cells were transfected with 0.5 mg/well of each of the following plasmids: pGL3-basic or various lengths of MMP13 promoter cloned into the pGL3-basic plasmid: 2660 bp, 2391 bp, 2391 bp DPEA, 291 bp, and 266 bp relative to transcription start site (generous gifts from Dr. Jeffrey M. Davidson, Vanderbilt University, Nashville and Dr. Keith Kirk- wood, University of Michigan, Ann Arbor). Cells were also co- transfected with the empty pCMV-HA vector (Clontech) or pCMV-HA-Ets1 (containing an HA-tagged version of mouse Ets1) and with pCMV-LacZ (0.25 mg/well) as an internal control. Cells were harvested 48 hours post transfection in Reporter Lysis buffer (Promega) and luciferase assays were performed. b-galactosidase values were measured using the Galacton Plus kit (Applied Biosystems). The luciferase values were normalized to b-galacto- sidase levels to correct for transfection efficiency. Reporter assays were performed in duplicates of at least three independent experiments and expressed as the means6S.D. Chromatin Immunoprecipitation Dorsal skins from wild-type and BT adult mice were de-epilated and treated with ammonium thiocyanate solution to separate the epidermis from the underlying dermis. The epidermal cells were disaggregated and fixed in 1% formaldehyde for 15 mins at RT. After washing, the cells were sonicated to fragment chromatin. The chromatin was then immunoprecipitated using the Chroma- tin Immunoprecipitation Assay kit (Upstate Biotechnology) and the following antibodies: anti-HA, anti-Ets1, isotype-control rat IgG or isotype control rabbit IgG. Immunoprecipitated samples were analyzed by PCR using primers designed to the minimal promoter of mouse MMP13: forward 59 TCCATTTCCCTCA- GATTCTGCCAC-39 and reverse 59-CAGCAGTGCCTG- GAGTCTCT-39. As a control, PCR was also performed using primers that recognize the minimal promoter of mouse GAPDH. Online Supplemental Material Hematoxylin and eosin staining and immuno-staining results presented in the online supplementary figures were collected using the same techniques and reagents as described above. Supporting Information Figure S1 Ets1 is not essential for epidermal development. (A) Ets1 knockout mouse demonstrating areas of white spotting (non- pigmented hair and skin) (B) Hematoxylin and eosin stained section of adult dorsal skin from an Ets1 knockout mouse, showing normal histology. C. High power view of boxed area in B. Found at: doi:10.1371/journal.pone.0004179.s001 (1.48 MB TIF) Figure S2 Loss of basement membrane integrity in re-induced lesions. Immunostaining for laminin I (red) shows breaks in basement membrane (arrows) in cutaneous lesions of the re- induced BT mice. Tissues are counterstained with K14 (to mark keratinocytes) and DAPI (to mark nuclei). Found at: doi:10.1371/journal.pone.0004179.s002 (1.87 MB TIF) Figure S3 Enhanced keratinocyte proliferation and block to terminal differentiation in the re-induced tumors. Detection of stage-specific keratinocyte differentiation and proliferation mark- ers in re-induced lesions by immunostaining (DAB staining, brown). The lesions display enhanced expression of early (keratin 5, K5) and intermediate differentiation (keratin 1, K1 and involucrin, inv) markers and decreased expression of the late differentiation marker loricrin (Lor). There is also enhanced PLoS ONE | www.plosone.org 13 January 2009 | Volume 4 | Issue 1 | e4179