felt to be involved in the pathogenesis of a large number of common malignancies. As market-driven forces generate means of targeting this pathway in common adult malignancies[8], the potential application of these means to patients with Ewing Sarcoma Family Tumors promises to improve outcomes in this difficult malignancy. Materials and Methods Retroviral Experiments The generation of recombinant retroviral stocks has been previously described [32] and in these experiments was modified only by using the LINX-A packaging line (Genetica) for retroviral constructs. These experiments employed either the vector system in that description or pLXIN (Clontech, Mountain View, CA) or the derivative pLXIH[4]. The retroviral construct for murine SHH was previously described [4]. The retroviral construct for Human Suppressor of Fused was obtained by PCR amplification of the reading frame from the clone MGC3533158 (Invitrogen). This insert was cloned into pLXIN (Clontech) or pLXIH [4]. Cloning strategy is available on request. RNAi experiments shRNA experiments were performed as previously described[4]. Target oligo sequences were obtained from the following publica- tions: EF2 [26], EF4 [26], EF818 [33]. Other shRNA oligo sequences can be found in Figure S1 accompanying this paper online. The antisense EWS/FLI1 construct and its use has been previously described [15]. Real Time Quantitative PCR (qPCR) RNA was isolated using either Tri-Reagent (MRC) or RNeasy Plus Kit (Qiagen, Valencia, CA) according to manufacturers instructions. CDNA’s were prepared from 0.5 mcg total RNA using the BioRad iScript Kit according to manufacturer’s instructions. PCR was performed using a BioRad MyiQ Thermal Cycler using BioRad iQ SYBR Green Supermix according to manufacturer instructions (BioRad, Hercules, CA). Conditions selected were 30 seconds of denaturation at 94uC and annealing/ extension for one minute at 60uC. Data was analyzed for expression relative to GAPDH using the comparative Ct method with data resulting from the average of three replica wells within each experiment. Data in figures is represented either as a percentage of GAPDH expression or the percent GAPDH values have been normalized with the control value set at 1.0. Results shown average the results of three independent experiments. Error bars show the standard error of the mean. P values were calculated using an unpaired Student’s t-Test. Primers for human GLI1, GLI2, Patched1, and GAPDH have been previously described[4]. Other primers sequences are in Figure S1 online. Cells, Cell Culture, and Materials All tumor cell lines were grown in RPMI supplemented with 2 mM glutamine and 10% Fetal Bovine Serum at 37 degrees C and in 5% CO2. TC71, TC32, TTC466, and TTC475 (plus primary specimen) were kindly provided by Dr. Tim Triche. References GLI1 in Ewing Tumors CHLA9 was provided by Dr. C. Patrick Reynolds. A673 cells were obtained directly from ATCC. Primary tumor specimens were obtained from Cooperative Human Tissue Network (CHTN) (http://www.chtn.nci.nih.gov). RMS-R and RMS-T RNA specimens were kindly provided by Dr. Michael Anderson. SaOS RNA was kindly provided by Dr. Daniel Wai. We appreciate their help in this regard. Drug Experiments Cells were exposed to Sonic Hedgehog C24II amino-terminal peptide (R&D Systems: http://www.rndsystems.com) for 16 hours before RNA was extracted. GANT58 and Forskolin (FSK) were obtained from EMD Biosciences (http://www.emdbiosciences.com, Cat #344270). Cyclopamine was obtained from Toronto Research Chemicals (http://www.trc-canada.com). Drugs were dissolved in DMSO. For expression experiments, cells were incubated with cells overnight. Antibodies and Immunoblotting These were performed as previously described[4]. Soft Agar Transformation Assay Polyclonal, selected populations of the cell type to be analyzed were plated in soft agar at either 5000 or 15000 cells per well of a 6-well plate. The agar was made with Iscove’s medium containing 20% fetal calf serum for tumor cell lines as described previously [32]. Drugs or diluent were added to the top layer of the agar in an amount to achieve the target concentration in the whole well. Drug was added only at the time of Agar setup. Agar plates were imaged via a transilluminating flatbed scanner approximately 2–3 weeks after plating. Counts were performed manually on high resolution scanned images. Results shown for these agar assays were consistent over at least three independent experiments. Lighter background color on some scanned agar images results from acidification of phenol red in the media, a sign of enhanced anchorage independent growth. Reproducibility Data presented has been repeated at least three times with consistent results. Numerical data presented is the average of these replicated experiments. Where necessary, results across experi- ments have been normalized to a control value. Error bars presented are the standard error of the mean. Supporting Information Figure S1 Oligonucleotide sequences Found at: doi:10.1371/journal.pone.0007608.s001 (0.00 RTF) MB Author Contributions Conceived and designed the experiments: ERL WAM. Performed the experiments: JJ LC KW LS HGK KV. Analyzed the data: WAM. Contributed reagents/materials/analysis tools: HGK ERL. Wrote the paper: WAM. 1. Arvand A, Denny CT (2001) Biology of EWS/ETS fusions in Ewing’s family tumors. Oncogene 20: 5747–5754. 2. Janknecht R (2005) EWS-ETS oncoproteins: the linchpins of Ewing tumors. Gene 363: 1–14. 3. Hancock JD, Lessnick SL (2008) A transcriptional profiling meta-analysis reveals a core EWS-FLI gene expression signature. Cell Cycle 7: 250–256. 4. Zwerner JP, Joo J, Warner KL, Christensen L, Hu-Lieskovan S, et al. (2008) The EWS/FLI1 oncogenic transcription factor deregulates GLI1. Oncogene 27: 3282–3291. 5. Beauchamp E, Bulut G, Abaan OD, Chen K, Merchant A, et al. (2009) GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein. J Biol Chem 284: 9074–9082. PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7608