immediately post treatment with inhibitors. For studying the effect of RhoGTPase activation, mMSCs were cultured for 8 hours on fibronectin, followed by overnight serum starvation and then treated with 0–100 mg/ml CP for 10 minutes or 0–100 ng/ml EGF for 2 minutes in serum free media. Subsequently, immedi- ately post treatment with activators, bolus transfection was done for 4 hours in serum free media. For assessing the role of ROCK and PKC in gene transfer, mMSCs were cultured for 16 hours on fibronectin prior to treatment with 10 mM Y27632 (Y2) for 2 hours or 100 nM Staurosporine (ST) for 2 hours to inhibit ROCK and PKC, respectively. Immediately after treatment with ST or Y2, the medium was replaced and bolus transfection was done. Cell proliferation was analyzed immediately after treatment with TcdB (A), C3 transferase (B), CP (C), EGF (D) and ST or Y2 (E), as well 48 hrs post treatment and transfection by measuring calcien AM fluorescence after live/dead staining. The cell viability obtained after specific inhibitor treatment were statistically compared using Tukey-Kramer Multiple Comparison test, which compares all pairs with each other. The symbols *, **, and *** represents a significant change to the level of p,0.05, p,0.01, and p,0.001 respectively. (TIF) Figure S2 Percent cells transfected with dominant negative and constitutively active genes, and the effect on cell viability. To study the effect of direct inhibition and activation of RhoGTPase on gene transfer in cells plated on fibronectin tissue culture plastic, D1 cells were transiently References 1. Li S, Huang L (2000) Nonviral gene therapy: promises and challenges. Gene Ther 7: 31–34. 2. Kircheis R, Wightman L, Wagner E (2001) Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev 53: 341–358. 3. Hassani Z, Francois JC, Alfama G, Dubois GM, Paris M, et al. (2007) A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain. Nucleic Acids Res 35: e65. 4. Kong HJ, Liu J, Riddle K, Matsumoto T, Leach K, et al. (2005) Non-viral gene delivery regulated by stiffness of cell adhesion substrates. Nat Mater 4: 460–464. 5. Uchimura E, Yamada S, Uebersax L, Yoshikawa T, Matsumoto K, et al. (2005) On-chip transfection of PC12 cells based on the rational understanding of the role of ECM molecules: efficient, non-viral transfection of PC12 cells using collagen IV. Neuroscience Letters 378: 40–43. 6. Dhaliwal A, Maldonado M, Han Z, Segura T (2010) Differential uptake of DNA-poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces. Acta Biomater 6: 3436–3447. 7. Bengali Z, Rea JC, Shea LD (2007) Gene expression and internalization following vector adsorption to immobilized proteins: dependence on protein identity and density. J Gene Med 9: 668–678. 8. Plautz SA, Boanca G, Riethoven JJ, Pannier AK (2011) Microarray analysis of gene expression profiles in cells transfected with nonviral vectors. Molecular therapy : the journal of the American Society of Gene Therapy 19: 2144–2151. 9. Perlstein I, Connolly JM, Cui X, Song C, Li Q, et al. (2003) DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid- controlled release coating: mechanisms of enhanced transfection. Gene Ther 10: 1420–1428. 10. Dhaliwal A, Lam J, Maldonado M, Lin C, Segura T (2011) Extracellular matrix modulates non-viral gene transfer to mouse mesenchymal stem cells. Soft Matter. 11. Arthur WT, Noren NK, Burridge K (2002) Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion. Biol Res 35: 239–246. 12. Morgan MR, Humphries MJ, Bass MD (2007) Synergistic control of cell adhesion by integrins and syndecans. Nat Rev Mol Cell Biol 8: 957–969. 13. Defilippi P, Olivo C, Venturino M, Dolce L, Silengo L, et al. (1999) Actin cytoskeleton organization in response to integrin-mediated adhesion. Microsc Res Tech 47: 67–78. 14. Price LS, Leng J, Schwartz MA, Bokoch GM (1998) Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell 9: 1863–1871. 15. Olson MF, Ashworth A, Hall A (1995) An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269: 1270–1272. 16. Schmidt A, Hall MN (1998) Signaling to the actin cytoskeleton. Annu Rev Cell Dev Biol 14: 305–338. 17. Nobes CD, Hall A (1999) Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144: 1235–1244. RhoGTPases and Non-Viral Gene Delivery transfected using lipofectamineTM2000, with dominant negative or constitutively active forms of RhoA, Rac1 or Cdc42 conjugated with GFP. The distribution and percentage of cells transfected with dominant negative genes (A) or constitutively active genes (B), was assessed by analyzing GFP expression using flowcytometry. The cells were subsequently cultured on Fn for 16 hours prior to bolus transfection using linear polyethyleneimine (LPEI). The cell viability was determined 16 hours after culturing cells on fibronectin as well as 48 hours post addition of polyplexes using live/dead assay, for cells transfected with dominant negative genes (C) or constitutively active genes (D). (TIF) Acknowledgments We sincerely appreciate the co-operation and valuable advice rendered by all the members of the Segura Lab, especially Yuguo Lei, Quinn Ng, Sean Anderson, Talar Tokatlian, Shiva Gojggini and Jonathan Lam. Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards CA- 16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA. Author Contributions Conceived and designed the experiments: TS AD. Performed the experiments: AD MM CL. Analyzed the data: AD TS. Wrote the paper: AD TS. 18. Kazmierczak BI, Jou TS, Mostov K, Engel JN (2001) Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells. Cell Microbiol 3: 85–98. 19. Burnham CA, Shokoples SE, Tyrrell GJ (2007) Rac1, RhoA, and Cdc42 participate in HeLa cell invasion by group B streptococcus. FEMS Microbiol Lett 272: 8–14. 20. Li E, Stupack D, Bokoch GM, Nemerow GR (1998) Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases. J Virol 72: 8806–8812. 21. Lamaze C, Chuang TH, Terlecky LJ, Bokoch GM, Schmid SL (1996) Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 382: 177–179. 22. Boudreau NJ, Jones PL (1999) Extracellular matrix and integrin signalling: the shape of things to come. Biochem J 339(Pt 3): 481–488. 23. Subauste MC, Von Herrath M, Benard V, Chamberlain CE, Chuang TH, et al. (2000) Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J Biol Chem 275: 9725–9733. 24. Kim SJ, Lee ZW, Kweon SM, Kim S, Ha KS (2002) Regulation of reactive oxygen species and stress fiber formation by calpeptin in Swiss 3T3 fibroblasts. Cell Signal 14: 205–210. 25. Schoenwaelder SM, Burridge K (1999) Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases. J Biol Chem 274: 14359–14367. 26. Ridley AJ, Hall A (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399. 27. Pack DW, Hoffman AS, Pun S, Stayton PS (2005) Design and development of polymers for gene delivery. Nat Rev Drug Discov 4: 581–593. 28. Tosello-Trampont AC, Nakada-Tsukui K, Ravichandran KS (2003) Engulf- ment of apoptotic cells is negatively regulated by Rho-mediated signaling. J Biol Chem 278: 49911–49919. 29. Ellis S, Mellor H (2000) Regulation of endocytic traffic by rho family GTPases. Trends Cell Biol 10: 85–88. 30. Ridley AJ (2001) Rho proteins: linking signaling with membrane trafficking. Traffic 2: 303–310. 31. Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E (1998) Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression. J Cell Biol 143: 267–276. 32. Ren XD, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP- binding protein Rho by cell adhesion and the cytoskeleton. Embo J 18: 578–585. 33. Aktories K, Just I (2005) Clostridial Rho-inhibiting protein toxins. Curr Top Microbiol Immunol 291: 113–145. 34. Genth H, Dreger SC, Huelsenbeck J, Just I (2008) Clostridium difficile toxins: more than mere inhibitors of Rho proteins. Int J Biochem Cell Biol 40: 592–597. PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35046