siRNA sequences targeting SMURF1. Author Contributions generate, mutated bases denoted by bold text) and 59-CCACAG- CAAACCCGGCGGTCTCCTCCACG-39. Fifty thousand cells were seeded per 60 mm plate, and 8 mg of plasmid was transfected by Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Two days after transfection, cells from each 60 mm plate were re-plated into two 10 cm plates and grown to confluence over 28 days. Visible foci were counted after methanol fixation and Giemsa staining. Assays were done as triplicate transfections, and repeated at least once with similar results. Supporting Information Table S1 (PDF) Figure S1 Hs700T cells display TGFb-induced growth inhibition. Hs700T cells were plated, and then 2 ng/ml TGFb (or vehicle control) added and cell proliferation/viability assayed (by WST-1) daily. Assays were done in triplicate (mean +/2 1SD References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249. 2. Li D, Xie K, Wolff R, Abbruzzese JL (2004) Pancreatic cancer. Lancet 363: 1049–1057. 3. Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA (2006) Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev 20: 1218–1249. 4. Hruban RH, Maitra A, Schulick R, Laheru D, Herman J, et al. (2008) Emerging molecular biology of pancreatic cancer. Gastrointest Cancer Res 2: S10–15. 5. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, et al. (2008) Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321: 1801–1806. 6. Kwei KA, Bashyam MD, Kao J, Ratheesh R, Reddy EC, et al. (2008) Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobil- iary cancer. PLoS Genet 4: e1000081. 7. Chen S, Auletta T, Dovirak O, Hutter C, Kuntz K, et al. (2008) Copy number alterations in pancreatic cancer identify recurrent PAK4 amplification. Cancer Biol Ther 7. 8. Aguirre AJ, Brennan C, Bailey G, Sinha R, Feng B, et al. (2004) High-resolution characterization of the pancreatic adenocarcinoma genome. Proc Natl Acad Sci U S A 101: 9067–9072. 9. Heidenblad M, Schoenmakers EF, Jonson T, Gorunova L, Veltman JA, et al. (2004) Genome-wide array-based comparative genomic hybridization reveals multiple amplification targets and novel homozygous deletions in pancreatic carcinoma cell lines. Cancer Res 64: 3052–3059. 10. Holzmann K, Kohlhammer H, Schwaenen C, Wessendorf S, Kestler HA, et al. (2004) Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes. Cancer Res 64: 4428–4433. 11. Mahlamaki EH, Kauraniemi P, Monni O, Wolf M, Hautaniemi S, et al. (2004) High-resolution genomic and expression profiling reveals 105 putative amplification target genes in pancreatic cancer. Neoplasia 6: 432–439. 12. Bashyam MD, Bair R, Kim YH, Wang P, Hernandez-Boussard T, et al. (2005) Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer. Neoplasia 7: 556–562. 13. Loukopoulos P, Shibata T, Katoh H, Kokubu A, Sakamoto M, et al. (2007) Genome-wide array-based comparative genomic hybridization analysis of pancreatic adenocarcinoma: identification of genetic indicators that predict patient outcome. Cancer Sci 98: 392–400. 14. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35: D61–65. 15. Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, et al. (2001) Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem 276: 12477–12480. 16. Moren A, Imamura T, Miyazono K, Heldin CH, Moustakas A (2005) Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases. J Biol Chem 280: 22115–22123. SMURF1 Amplification in Pancreatic Cancer shown); *, P,0.05; **, P,0.01; ***, P,0.001 (Student’s t-test). Consistent with intact TGFb growth inhibition, no deletions spanning SMAD4 (244K Agilent CGH array data; not shown) and no point mutations of SMAD4 (Illumina RNAseq analysis; not shown) were identified. (EPS) Acknowledgments We thank Ilana Galperin (Stanford Cytogenetics Laboratory) for assistance with FISH analysis, and the members of the Pollack lab for helpful discussions. Conceived and designed the experiments: KAK AHS JRP. Performed the experiments: KAK AHS RB MDB. Analyzed the data: KAK AHS JRP. Contributed reagents/materials/analysis tools: KM CAK MvdR MH AM. Wrote the paper: KAK AHS JRP. 17. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, et al. (1992) TGF beta signals through a heteromeric protein kinase receptor complex. Cell 71: 1003–1014. 18. Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, et al. (1996) DPC4 gene in various tumor types. Cancer Res 56: 2527–2530. 19. Dai JL, Turnacioglu KK, Schutte M, Sugar AY, Kern SE (1998) Dpc4 transcriptional activation and dysfunction in cancer cells. Cancer Res 58: 4592–4597. 20. Wan M, Huang J, Jhala NC, Tytler EM, Yang L, et al. (2005) SCF(beta-TrCP1) controls Smad4 protein stability in pancreatic cancer cells. Am J Pathol 166: 1379–1392. 21. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, et al. (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271: 350–353. 22. Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, et al. (2003) Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302: 1775–1779. 23. Massague J (2008) TGFbeta in Cancer. Cell 134: 215–230. 24. Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y, et al. (2005) Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science 307: 1603–1609. 25. Huang C, Rajfur Z, Yousefi N, Chen Z, Jacobson K, et al. (2009) Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration. Nat Cell Biol 11: 624–630. 26. Suzuki A, Shibata T, Shimada Y, Murakami Y, Horii A, et al. (2008) Identification of SMURF1 as a possible target for 7q21.3-22.1 amplification detected in a pancreatic cancer cell line by in-house array-based comparative genomic hybridization. Cancer Sci 99: 986–994. 27. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5: 834–839. 28. Laurila E, Savinainen K, Kuuselo R, Karhu R, Kallioniemi A (2009) Characterization of the 7q21-q22 amplicon identifies ARPC1A, a subunit of the Arp2/3 complex, as a regulator of cell migration and invasion in pancreatic cancer. Genes Chromosomes Cancer 48: 330–339. 29. Kao J, Pollack JR (2006) RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes. Genes Chromosomes Cancer 45: 761–769. 30. Chen C, Matesic LE (2007) The Nedd4-like family of E3 ubiquitin ligases and cancer. Cancer Metastasis Rev 26: 587–604. 31. Guedat P, Colland F (2007) Patented small molecule inhibitors in the ubiquitin proteasome system. BMC Biochem 8 Suppl 1: S14. 32. Hahn SA, Seymour AB, Hoque AT, Schutte M, da Costa LT, et al. (1995) Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer Res 55: 4670–4675. 33. Pollack JR, Sorlie T, Perou CM, Rees CA, Jeffrey SS, et al. (2002) Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors. Proc Natl Acad Sci U S A 99: 12963–12968. 34. Tibshirani R, Wang P (2008) Spatial smoothing and hot spot detection for CGH data using the fused lasso. Biostatistics 9: 18–29. 35. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, et al. (2000) Molecular portraits of human breast tumours. Nature 406: 747–752. PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23924