Statistical analysis Differences between locostatin treated or untreated cells were compared using a two-tailed unpaired Student’s t-test. Supporting Information Figure S1 Locostatin disrupts the mitotic spindle and chromo- some organization. MEFs expressing (A) wild-type (RKIP+/+) or lacking RKIP (RKIP depleted or RKIP2/2) were plated at 26104 cells/coverslip and grown for 48 h before treatment with either DMSO (Control) or locostatin (20 mM) for either 1 or 2 h. Data represents the mean6S.E. percent abnormal mitotic spindles (n = 3), cells counted ranged from 33–119 for the various cell lines. References 1. Zeng L, Imamoto A, Rosner MR (2008) Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target. Expert Opin Ther Targets 12: 1275–1287. 2. Granovsky AE, Rosner MR (2008) Raf kinase inhibitory protein: a signal transduction modulator and metastasis suppressor. Cell Res 18: 452–457. 3. Yeung K, Seitz T, Li S, Janosch P, McFerran B, et al. (1999) Suppression of Raf- 1 kinase activity and MAP kinase signalling by RKIP. Nature 401: 173–177. 4. Corbit KC, Trakul N, Eves EM, Diaz B, Marshall M, et al. (2003) Activation of Raf-1 Signaling by Protein Kinase C through a Mechanism Involving Raf Kinase Inhibitory Protein. J Biol Chem 278: 13061–13068. 5. Trakul N, Menard RE, Schade GR, Qian Z, Rosner MR (2005) Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation. J Biol Chem 280: 24931–24940. 6. Lorenz K, Lohse MJ, Quitterer U (2003) Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426: 574–579. 7. Yeung KC, Rose DW, Dhillon AS, Yaros D, Gustafsson M, et al. (2001) Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation. Mol Cell Biol 21: 7207–7217. 8. Eves EM, Shapiro P, Naik K, Klein UR, Trakul N, et al. (2006) Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint. Mol Cell 23: 561–574. 9. Fu Z, Smith PC, Zhang L, Rubin MA, Dunn RL, et al. (2003) Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis. J Natl Cancer Inst 95: 878–889. 10. Dangi-Garimella S, Yun J, Eves EM, Newman M, Erkeland SJ, et al. (2009) Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J. 11. Banfield MJ, Barker JJ, Perry AC, Brady RL (1998) Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction. Structure 6: 1245–1254. 12. Granovsky AE, Clark MC, McElheny D, Heil G, Hong J, et al. (2009) Raf Kinase Inhibitory Protein Function is Regulated via Flexible Pocket and Novel Phosphorylation-dependent Mechanism. Mol Cell Biol. 13. Zhu S, Mc Henry KT, Lane WS, Fenteany G (2005) A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration. Chem Biol 12: 981–991. Locostatin Impairs Migration Found at: doi:10.1371/journal.pone.0006028.s001 (0.04 MB TIF) Acknowledgments We would like to thank Dr. Surabhi Dangi-Garimella for her initial observations of the effect of locostatin on EGF-induced MAPK activity. Author Contributions Conceived and designed the experiments: ANS MCC. Performed the experiments: ANS EME MCC GH AG. Analyzed the data: ANS EME MCC GH AG SK. Contributed reagents/materials/analysis tools: LZ AI. Wrote the paper: ANS SK. Conceived experiments and co-wrote manuscript: MRR. Assisted with writing and analyzing NMR data: SK. 14. Mc Henry KT, Montesano R, Zhu S, Beshir AB, Tang HH, et al. (2008) Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion. J Cell Biochem 103: 972–985. 15. Shuker SB, Hajduk PJ, Meadows RP, Fesik SW (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274: 1531–1534. 16. Clark MC, McElheny D, Wojcik J, Kurutz J, Rosner MR, et al. (2006) NMR assignment of rat Raf kinase inhibitor protein. J Biomol NMR 36 Suppl 1: 4. 17. Mc Henry KT, Ankala SV, Ghosh AK, Fenteany G (2002) A non-antibacterial oxazolidinone derivative that inhibits epithelial cell sheet migration. Chembio- chem 3: 1105–1111. 18. Schuierer MM, Heilmeier U, Boettcher A, Ugocsai P, Bosserhoff AK, et al. (2006) Induction of Raf kinase inhibitor protein contributes to macrophage differentiation. Biochem Biophys Res Commun 342: 1083–1087. 19. Lee HC, Tian B, Sedivy JM, Wands JR, Kim M (2006) Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells. Gastroenterology 131: 1208–1217. 20. Ma S, Chan KW, Lee TK, Tang KH, Wo JY, et al. (2008) Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res 6: 1146–1153. 21. Kuo PH, Kalsi G, Prescott CA, Hodgkinson CA, Goldman D, et al. (2008) Association of ADH and ALDH genes with alcohol dependence in the Irish Affected Sib Pair Study of alcohol dependence (IASPSAD) sample. Alcohol Clin Exp Res 32: 785–795. 22. Theroux S, Pereira M, Casten KS, Burwell RD, Yeung KC, et al. (2007) Raf kinase inhibitory protein knockout mice: expression in the brain and olfaction deficit. Brain Res Bull 71: 559–567. 23. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, et al. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6: 277–293. 24. Johnson BA, Blevins RA (1994) NMRView: A computer program for the visualization and analysis of NMR data. J Biomol NMR 4: 603–614. 25. Farmer BT 2nd, Constantine KL, Goldfarb V, Friedrichs MS, Wittekind M, et al. (1996) Localizing the NADP+ binding site on the MurB enzyme by NMR. Nat Struct Biol 3: 995–997. PLoS ONE | www.plosone.org 13 June 2009 | Volume 4 | Issue 6 | e6028