syndecans [12], which effectively engage RhoGTPases leading to adhesion signaling [13,14]. Rho proteins alternate between an active GTP-bound state and an inactive GDP-bound state. In the active conformation, GTPases interact with and stimulate the activity of effectors which participate in signaling cascades that coordinate various cellular processes such as migration, prolifer- ation [15], gene expression [16] and cytoskeletal organization. Studies have shown that Cdc42 mediates cell polarity and filipodia, Rac mediates protrusion of lamellipodia, and Rho maintains cell adhesion during migration [17]. Furthermore, bacterial internalization [18,19], adenovirus internalization [20] and recently receptor mediated internalization of transferrin [21] has been shown to be a resultant of host cell actin cytoskeleton manipulation at level of RhoGTPases. However, the role of RhoGTPases in non-viral gene transfer has not been previously investigated. It is likely that the interplay between Fn and integrins is communicated via the RhoGTPases [22] and the resultant signaling cascades affecting cytoskeletal dynamics, endocytosis, gene transcription and proliferation are regulating non-viral gene transfer in cells plated on Fn. Herein, to begin to understand the cellular molecules involved in modulating gene delivery to mMSCs plated on Fn coated surfaces, the role of Rho, Rac and Cdc42 RhoGTPases in the uptake and transgene expression of DNA/PEI polyplexes was studied. The RhoGTPases were specifically inhibited using chemical inhibitors and dominant negative gene products, as well as activated to determine their role in gene transfer and DNA/PEI internalization. Results Fibronectin exposure activates RhoGTPases in mMSCs The activation of RhoGTPases in mMSCs by fibronectin was assed using fluorescence immunohistochemistry and molecular assays. Fluorescence immunohistochemistry of the actin cytoskel- eton revealed that cells plated on fibronectin-coated surfaces resulted in an increase in stress fibers compared to cells plated on tissue culture plastic (Fig. 1A, B, p,0.05), indicative of active RhoGTPases. The level of RhoGTPase activation was determined using GLISA assays for RhoA, Rac1,2,3 and Cdc42. To ensure that the observed activation was due to the interaction of the cell with the fibronectin coated surface and not the dynamics of cell spreading, cells were allowed to attach and spread on amine functionalized PDMS sheets. Following cell spreading, the cell sheet was flipped on top of fibronectin coated or uncoated tissue culture plastic and incubated for 20 minutes or 1-hour. At 20 minutes of exposure to fibronectin-coated surfaces, RhoA was significantly activated (p,0.05), while Cdc42 was not activated and Rac1,2,3 was inhibited compared to exposure to bare tissue culture plastic (Fig. 1C). After 1-hour exposure to fibronectin- coated surfaces, Rac1,2,3 and Cdc42 were significantly activated (p,0.05), while RhoA was not activated (Fig. 1D). Inactivation of RhoGTPases for cells plated on fibronectin inhibits transgene expression Inactivation of RhoGTPases was used to screen their potential involvement in mediating non-viral gene transfer in mMSCs. Inactivation of RhoGTPase using difficile toxin B was character- ized using actin staining and GLISAs for RhoGTPases. Cells with inactivated RhoGTPases resulted in less spread cells with a significant decrease in stress fibers (p,0.01, Fig. 2A, B) and amount active RhoA, Rac1,2,3 and Cdc42 (p at least,0.05, Fig. 2C). Cell viability was not significantly affected immediately after treatment with TcdB and 48 hrs post transfection (Fig. S1A). RhoA,B,C were selectively inactivated using C3 transferase and RhoGTPases and Non-Viral Gene Delivery resulted in a significant decrease in actin stress fibers (p,0.01, Fig. 2A, B) and a significant decrease in active RhoA (p,0.01, Fig. 2D). Cell viability was maintained at .60% immediately post C3 transferase treatment as well as 48 hours after treatment and transfection (Fig. S1B). Cells were transfected immediately following the inhibitory treatments and transgene expression was analyzed using a luciferase assay at 48 hours and the extent of polyplex internal- ization was assessed using flow cytometry analysis (FACS) after 2- hours. More than 90% decrease in transgene expression of cells plated on fibronectin-coated surfaces was observed after treatment with TcdB and C3 transferase (p,0.001, Fig. 2E). However, internalization of the complexes was significantly increased following treatment (p at least,0.05, Fig. 2F). To further understand the individual role of RhoGTPases, mMSCs were transiently transfected with GFP fused plasmid constructs of dominant negative mutants of either RhoA (RhoA- T19N), or Rac1 (Rac1-T17N) or Cdc42 (Cdc42-T17N) [19,23]. At least 80% cell viability was maintained in cells expressing EGFP control plasmid and dominant negative genes 16-hours post plating cells on fibronectin (at time of bolus transfection) and 48 hrs post gene delivery (Fig. S2C). Expression of RhoA-T19N by cells plated on fibronectin coated surface resulted in a significant decrease (p,0.01) in stress fibers; however, no significant difference in stress fibers was observed in cells expressing dominant negative mutants of Rac1 and Cdc42 (Fig. 3A, B). GLISA analysis of active Rho, Rac1,2,3 and Cdc42 showed a significant decrease in active RhoA upon transfection with RhoA-T19N (p,0.05), but no change in active Rac or Cdc42 upon transfection with Rac1-T17N or Cdc42-T17N compared to cells transfected with EGFP (Fig. 3C). Transgene expression was significantly reduced in the cells expressing dominant negative form of RhoA, Rac1 and Cdc42 (p,0.05, Fig. 3D). The effect of dominant negative mutants on internalization was studied by analyzing the uptake of YOYO-3 labeled complexes in GFP positive cells only. A significant decrease in internalization was observed for cells expressing dominant negative form of RhoGTPases (p,0.01, Fig. 3E). Activation of RhoGTPases for cells plated on fibronectin does not influence transgene expression Since RhoGTPase inactivation lead to a significant decrease in gene transfer efficiency for cells plated on fibronectin-coated surfaces, next we wanted to determine if further activation of RhoGTPases enhanced transgene expression. Calpeptin (CP) was used to activate RhoA [24,25]. Treatment with CP did not significantly change the number of stress fibers in the cell (Fig. 4 A, B), but did significantly increased the amount of active Rho (by 2.5-fold) (p,0.01, Fig. 4C) compared to cells seeded on fibronectin coated surfaces that did not receive treatment. Viability was maintained at 90% or greater compared to cells not treated with CP immediately after treatment as well as 48 hours post treatment and addition of polyplexes (Fig. S1C). Epidermal growth factor (EGF) is an efficient activator of Rac1,2, and Cdc42 [26]. Treatment with EGF resulted in a significant increase in active Rac1,2,3 and Cdc42 (p,0.01 and ,0.01, Fig. 4C) compared to cells seeded on fibronectin-coated surfaces that did not receive treatment. No significant change in cell viability was observed immediately post treatment and 48 hours post treatment and transfection (Fig. S1D). No significant difference in transgene expression (Fig. 4E) and internalization (Fig. 4F) was observed using treatment with CP or EGF for cells placed on fibronectin. To confirm the CP and EGF findings, mMSCs were transiently transfected with GFP fused plasmid constructs of constitutive PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35046