Figure S4 Proliferation not affected by myoferlin de- pletion in MDA-MB-231 cells. Growth curves (A) and cell cycle analysis (B) of wild type (WT), lentiviral control (LTV-ctrl) and myoferlin knockdown (MYOF-KD) MDA-MB-231 cells. Statistical analysis on the proliferation curves (n $4, mean 6 s.d.) were done using linear regression on the log transformation of the OD readings (p = 0.43), showing an insignificance difference among the proliferation rate of the cells. The cell cycle analysis (n $4, mean 6 s.d.) also showed an insignificant difference in cell proliferation (Gaussian approximation P value of 0.96, Kruskal- Wallis/Dunn’s multiple comparison post-test). (TIF) Figure S5 Myoferlin depletion in MDA-MB-231 cells alters the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Anti- body membrane-based array detecting various MMPs and TIMPs was used to screen whether myoferlin depletion changes the secretion of MMPs and TIMPs. 231LTV-ctrl and 231MYOF-KD cells were serum starved for 24 h, and culture supernatants collected and analyzed for extracellular matrix proteins. The intensity of the quantified signals was normalized to 231LTV-ctrl cells, and results are expressed as fold changes. (TIF) Figure S6 MMP1 mRNA expression attenuated by myoferlin depletion in MDA-MB-231 cells. Relative levels of MMP1 mRNA in lentiviral-control (LTV-ctrl) and myoferlin depleted (MYOF-KD) MDA-MB-231 cells. Levels of 18 S expression were used to normalize the samples. Graph represents fold change normalized to MMP1 levels in 231LTV-ctrl cells (n = 3, mean 6 s.d.), showing a significant depletion of MMP1 mRNA in 231MYOF-KD cells (unpaired t-test, p = 0.003). (TIF) Figure S7 Myoferlin depletion in MDA-MB-231 cells by separate shRNA constructs attenuates MMP1 produc- tion. Immunoblotting (A) and ELISA (B) evaluation of secreted References 1. American Cancer Society (2009) Breast Cancer Facts & Figures 2009–2010. 2. Horner MRL, Krapcho M, Neyman N, Aminou R, Howlader N, et al. (2009) SEER Cancer Statistics Review, 1975–2006. National Cancer Institute. National Cancer Institute Surveillance Epidemiology and End Results website. Available: http://seer.cancer.gov/csr/1975_2006/. Accessed 2012 Jan 12. 3. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57–70. 4. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 5. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139: 871–890. 6. Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331: 1559–1564. 7. Sabeh F, Shimizu-Hirota R, Weiss SJ (2009) Protease-dependent versus - independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. J Cell Biol 185: 11–19. 8. Yilmaz M, Christofori G (2010) Mechanisms of motility in metastasizing cells. Mol Cancer Res 8: 629–642. 9. Fletcher SJ, Rappoport JZ (2009) The role of vesicle trafficking in epithelial cell motility. Biochem Soc Trans 37: 1072–1076. 10. Caswell P, Norman J (2008) Endocytic transport of integrins during cell migration and invasion. Trends Cell Biol 18: 257–263. 11. Mills GB, Jurisica I, Yarden Y, Norman JC (2009) Genomic amplicons target vesicle recycling in breast cancer. J Clin Invest 119: 2123–2127. 12. Frittoli E, Palamidessi A, Disanza A, Scita G (2011) Secretory and endo/ exocytic trafficking in invadopodia formation: the MT1-MMP paradigm. Eur J Cell Biol 90: 108–114. 13. Caldieri G, Buccione R (2010) Aiming for invadopodia: organizing polarized delivery at sites of invasion. Trends Cell Biol 20: 64–70. 14. Lek A, Lek M, North KN, Cooper ST (2010) Phylogenetic analysis of ferlin genes reveals ancient eukaryotic origins. BMC Evol Biol 10: 231. 15. Washington NL, Ward S (2006) FER-1 regulates Ca2+ -mediated membrane fusion during C. elegans spermatogenesis. J Cell Sci 119: 2552–2562. Myoferlin in Breast Cancer EMT & Invasion MMP1 in 24 h serum starved supernatant of myoferlin depleted MDA-MB-231 cells (constructs #10628 and #320398). Three replicate samples of construct #320398 were ran (A–C). ELISA results (n = 3, mean 6 s.d.) show significant depletion of secreted MMP1 in both myoferlin depleted MDA-MB-231 cells (one way ANOVA/Tukey’s Multiple Comparison Test, p,0.01) when compared with the lentiviral control cells. (TIF) Table S1 Reported is an alphabetical listing of genes with Cp values less than 30 cycles for at least one of the samples and with fold changes of $ 62-fold. Fold change for genes up-regulated in MYOF-deficient MDA-MB-231 cells are in bold, while the down-regulated genes are in italics. (DOCX) Table S2 Reported are fold changes at the mRNA level, along with each MMP’s known ECM substrates [54]. MMPs with Cp values less than 30 cycles for at least one of the samples are formatted in bold (MMP1, MMP10, MMP11, and MMP14). Fold change for genes down-regulated in MYOF- deficient MDA-MB-231 cells are in italics. (DOCX) Acknowledgments The technical expertise of Taryn Summerfield is gratefully acknowledged. We also thank the Campus Microscopy and Imaging Facility and the Atomic Force Microscopy Core at The Ohio State University for assistance with confocal microscopy, SEM and AFM. The authors thank Drs. Tom Rosol and Matthew Ringel for critically reading of the manuscript as it was first prepared. Author Contributions Conceived and designed the experiments: DAK WEA RL SG. Performed the experiments: RL CM LIV WEA. Analyzed the data: RL WEA CM LIV DAK. Contributed reagents/materials/analysis tools: DAK WEA SG. Wrote the paper: RL WEA CM LIV SG DAK. 16. Liu J, Aoki M, Illa I, Wu C, Fardeau M, et al. (1998) Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 20: 31–36. 17. Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, et al. (2003) Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound- healing. J Biol Chem 278: 50466–50473. 18. Bansal D, Campbell KP (2004) Dysferlin and the plasma membrane repair in muscular dystrophy. Trends Cell Biol 14: 206–213. 19. Han R, Campbell KP (2007) Dysferlin and muscle membrane repair. Curr Opin Cell Biol 19: 409–416. 20. Yasunaga S, Grati M, Chardenoux S, Smith TN, Friedman TB, et al. (2000) OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9. Am J Hum Genet 67: 591–600. 21. Roux I, Safieddine S, Nouvian R, Grati M, Simmler MC, et al. (2006) Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell 127: 277–289. 22. Doherty KR, Cave A, Davis DB, Delmonte AJ, Posey A,et al. (2005) Normal myoblast fusion requires myoferlin. Development 132: 5565–5575. 23. Bernatchez PN, Sharma A, Kodaman P, Sessa WC (2009) Myoferlin is critical for endocytosis in endothelial cells. Am J Physiol Cell Physiol 297: C484–C492. 24. Bernatchez PN, Acevedo L, Fernandez-Hernando C, Murata T, Chalouni C, et al. (2007) Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function. J Biol Chem 282: 30745–30753. 25. Yu C, Sharma A, Trane A, Utokaparch S, Leung C, et al. (2011) Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol. 26. Demonbreun AR, Posey AD, Heretis K, Swaggart KA, Earley JU, et al. (2010) Myoferlin is required for insulin-like growth factor response and muscle growth. FASEB J 24: 1284–1295. 27. van ’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, et al. (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415: 530– 536. PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39766