were stained with Rhodamine phalloidin (Molecular probes) and F-actin cytoskeleton was viewed under an Olympus Fluoview FV1000 confocal fluorescence microscope. Found at: doi:10.1371/journal.pone.0010365.s003 (0.28 MB TIF) Figure S4 Prolonged treatment of 4T1 cells with TGFb1 enhanced formation of mammospheres. 4T1 cells were treated with 0.05 or 0.1 ng/ml of TGFb1 for 12 days with medium change every alternate day. Cells were harvested and passed through a 40 mm nylon membrane to obtain a single cell suspension. Cells were plated (2,000 cells per well) in a 24-well ultra low attachment plate in DMEM-F12 mammosphere culture medium. The number of large mammospheres (.100 mm in diameter) obtained from 2,000 cells with or without TGFb1 References 1. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108. 2. Gamucci T, D’Ottavio AM, Magnolfi E, Barduagni M, Vaccaro A, et al. (2007) Weekly epirubicin plus docetaxel as first-line treatment in metastatic breast cancer. Br J Cancer 97: 1040–1045. 3. Sledge GW, Neuberg D, Bernardo P, Ingle JN, Martino S, et al. (2003) Phase III trial of doxorubicin, paclitaxel, and the combination of doxorubicin and paclitaxel as front-line chemotherapy for metastatic breast cancer: an intergroup trial (E1193). J Clin Oncol 21: 588–592. 4. Ellis MJ, Hayes DF, Lippman ML (2004) Treatment of metastatic breast cancer: Diseases of the breast 3rd: 749–797. 5. Chen JH, Ling R, Yao Q, Li Y, Chen T, et al. (2005) Effect of small-sized liposomal Adriamycin administered by various routes on a metastatic breast cancer model. Endocr Relat Cancer 12: 93–100. 6. Gorsch SM, Memoli VA, Stukel TA, Gold LI, Arrick BA (1992) Immunohis- tochemical staining for transforming growth factor beta 1 associates with disease progression in human breast cancer. Cancer Res 52: 6949–6952. 7. Walker RA, Gallacher B (1995) Determination of transforming growth factor beta 1 mRNA expression in breast carcinomas by in situ hybridization. J Pathol 177: 123–127. 8. Tang BW, Vu M, Booker T, Santner SJ, Miller FR, et al. (2003) TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression. Journal of Clinical Investigation 112: 1116–1124. 9. Ivanovic V, Todorovic-Rakovic N, Demajo M, Neskovic-Konstantinovic Z, Subota V, et al. (2003) Elevated plasma levels of transforming growth factor- beta(1) (TGF-beta(1)) in patients with advanced breast cancer: association with disease progression. European Journal of Cancer 39: 454–461. 10. Dalal BI, Keown PA, Greenberg AH (1993) Immunocytochemical localization of secreted transforming growth factor-beta 1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma. Am J Pathol 143: 381–389. 11. Verona EV, Elkahloun AG, Yang J, Bandyopadhyay A, Yeh IT, et al. (2007) Transforming growth factor-beta signaling in prostate stromal cells supports prostate carcinoma growth by up-regulating stromal genes related to tissue remodeling. Cancer Res 67: 5737–5746. 12. Wikstro¨m P, Stattin P, Franck-Lissbrant I, Damber JE, Bergh A (1998) Transforming growth factor b1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer. Prostate 37: 19–29. 13. Sosroseno W, Herminajeng E (1995) The immunoregulatory roles of transforming growth factor beta. Br J Biomed Sci 52: 142–148. 14. Lei XF, Bandyopadhyay A, Le T, Sun LZ (2002) Autocrine TGF beta supports growth and survival of human breast cancer MDA-MB-231 cells. Oncogene 21: 7514–7523. 15. Lei X, Yang J, Nichols RW, Sun LZ (2007) Abrogation of TGFbeta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells. Exp Cell Res 313: 1687–1695. 16. Arteaga CL (2006) Inhibition of TGFbeta signaling in cancer therapy. Curr Opin Genet Dev 16: 30–37. 17. Sun L (2004) Tumor-suppressive and promoting function of transforming growth factor beta. Front Biosci 9: 1925–1935. 18. Yingling JM, Blanchard KL, Sawyer JS (2004) Development of TGF-beta signalling inhibitors for cancer therapy. Nat Rev Drug Discov 3: 1011–1022. 19. Bandyopadhyay A, Sun L-Z (2008) Soluble TGF beta Type III Receptor Suppresses Malignant Progression of Human Cancer Cells. Cancer Drug Discovery and Development: Transforming Growth Factor-beta in Cancer Therapy, Vol II: Cancer Treatment and Therapy. pp 723–735. 20. Muraoka-Cook RS, Shin I, Yi JY, Easterly E, Barcellos-Hoff MH, et al. (2006) Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression. Oncogene 25: 3408–3423. 21. Ehata S, Hanyu A, Fujime M, Katsuno Y, Fukunaga E, et al. (2007) Ki26894, a novel transforming growth factor-beta type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line. Cancer Sci 98: 127–133. Doxorubicin and TGFb Inhibitor treatment were counted and presented in Panel A. The representative photos of mammosphere are shown in Panel B. Found at: doi:10.1371/journal.pone.0010365.s004 (0.10 MB TIF) Acknowledgments The authors thank the institutional cores of FACS, Optical Imaging, and Pathology for their assistance in obtaining part of the presented data. Author Contributions Conceived and designed the experiments: AB LZS. Performed the experiments: AB LW YT SL KD. Analyzed the data: AB LW YT SL KD. Contributed reagents/materials/analysis tools: JA. Wrote the paper: AB LZS. Histology analysis: ITY. 22. Bandyopadhyay A, Agyin JK, Wang L, Tang Y, Lei X, et al. (2006) Inhibition of Pulmonary and Skeletal Metastasis by a Transforming Growth Factor-{beta} Type I Receptor Kinase Inhibitor. Cancer Res 66: 6714–6721. 23. Ge R, Rajeev V, Ray P, Lattime E, Rittling S, et al. (2006) Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transforming growth factor-beta type I receptor kinase in vivo. Clin Cancer Res 12: 4315–4330. 24. Suzuki E, Kim S, Cheung HK, Corbley MJ, Zhang X, et al. (2007) A Novel Small-Molecule Inhibitor of Transforming Growth Factor {beta} Type I Receptor Kinase (SM16) Inhibits Murine Mesothelioma Tumor Growth In vivo and Prevents Tumor Recurrence after Surgical Resection. Cancer Res 67: 2351–2359. 25. Ehata S, Hanyu A, Hayashi M, Aburatani H, Kato Y, et al. (2007) Transforming Growth Factor-{beta} Promotes Survival of Mammary Carcinoma Cells through Induction of Antiapoptotic Transcription Factor DEC1. Cancer Res 67: 9694–9703. 26. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, et al. (2008) The epithelial- mesenchymal transition generates cells with properties of stem cells. Cell 133: 704–715. 27. Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, et al. (2008) Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS ONE 3: e2888. 28. Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, et al. (2007) Molecular definition of breast tumor heterogeneity. Cancer Cell 11: 259–273. 29. Teicher BA, Kakeji Y, Ara G, Herbst RS, Northey D (1997) Prostate carcinoma response to cytotoxic therapy: in vivo resistance. In Vivo 11: 453–461. 30. Liu P, Menon K, Alvarez E, Lu K, Teicher BA (2000) Transforming growth factor-beta and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo. Int J Oncol 16: 599–610. 31. Teicher BA, Ikebe M, Ara G, Keyes SR, Herbst RS (1997) Transforming growth factor-beta 1 overexpression produces drug resistance in vivo: reversal by decorin. In Vivo 11: 463–472. 32. Biswas S, Guix M, Rinehart C, Dugger TC, Chytil A, et al. (2007) Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression. J Clin Invest 117: 1305–1313. 33. Hollier BG, Evans K, Mani SA (2009) The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies. J Mammary Gland Biol Neoplasia 14: 29–43. 34. Wu Y, Zhou BP (2008) New insights of epithelial-mesenchymal transition in cancer metastasis. Acta Biochim Biophys Sin (Shanghai) 40: 643–650. 35. Xu J, Lamouille S, Derynck R (2009) TGF-beta-induced epithelial to mesenchymal transition. Cell Res 19: 156–172. 36. Medici D, Hay ED, Goodenough DA (2006) Cooperation between snail and LEF-1 transcription factors is essential for TGF-beta1-induced epithelial- mesenchymal transition. Mol Biol Cell 17: 1871–1879. 37. Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, et al. (2002) Sca- 1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 245: 42–56. 38. Dean M (2009) ABC transporters, drug resistance, and cancer stem cells. J Mammary Gland Biol Neoplasia 14: 3–9. 39. Dontu G, Wicha MS (2005) Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia 10: 75–86. 40. Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, et al. (2008) Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 10: R52. 41. Yoneda T, Michigami T, Yi B, Williams PJ, Niewolna M, et al. (2000) Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma. Cancer 88: 2979–2988. 42. Nikolic-Vukosavljevic D, Todorovic-Rakovic N, Demajo M, Ivanovic V, Neskovic B, et al. (2004) Plasma TGF-beta1-related survival of postmenopausal metastatic breast cancer patients. Clin Exp Metastasis 21: 581–585. PLoS ONE | www.plosone.org 12 April 2010 | Volume 5 | Issue 4 | e10365