Figure 5. Effect of expression of constitutively active mutants of RhoGTPases on gene transfer. mMSCs plated on a 6-well plate were transiently transfected with constitutive active forms of RhoA, Rac1 or Cdc42 conjugated with GFP, using lipofectamineTM2000. Cells transfected with pEGFP were used as control. After 24 hours, cells were harvested and replated on fibronectin-coated surfaces. (A) 24 hours post transfection with constitutive active forms of RhoGTPases in cells plated on tissue culture plastic, changes in cell morphology were analyzed through actin and DNA staining using Alexa488 conjugated phalloidin (green), and Hoechst 33258 dye (blue), respectively. (B) The number of stress fibers per cell were counted. (C) Active Rho, Rac and Cdc42-GTPases were assessed using GLISA assays 24 hours post transfection with constitutive active forms of RhoGTPases and overnight serum starvation. (D) The replated cells were cultured for 16 hours on fibronectin before bolus transfection. The transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. (E) Internalization was analyzed 2 hours post transfection using flow cytometry and YOYO-3 labeled polyplexes. A total of 10,000 events were analyzed per sample and the mean fluorescence of events positive for both GFP and YOYO-3 was analyzed. Statistical analysis for number of stress fibers, ruffles, transgene expression and internalization was done using the Dunnett multiple comparison test. doi:10.1371/journal.pone.0035046.g005 mediate cell spreading in NIH/3T3 cells [14,32]. Our results show that exposure of mMSCs to fibronectin coated surfaces results in enhanced activation of the RhoGTPases namely RhoA, Rac1,2,2 RhoGTPases and Non-Viral Gene Delivery and Cdc42, in mMSCs. To study the role of RhoGTPases on non- viral gene transfer for mMSCs plated on fibronectin coated surfaces, systematic inhibition and activation by molecular inhibitors as well as dominant negative or positive genes were used. RhoGTPases were inhibited using bacterial toxins (TcdB and C3 transferase) and dominant negative genes. TcdB irreversibly inactivates Rho, Rac and Cdc42 by glycosylation of the threonine residue [33,34], while C3 selectively inhibits RhoA, B and C [35], but not Rac or Cdc42, by modifying the aspargine41 at the effector region of the GTPase. Inhibition by bacterial toxins resulted in a significant decrease (at least p,0.05) in the overall amount of active Rho, Rac and Cdc42. Further, a significant decrease (p,0.05) was observed in active RhoA upon transfection with RhoAT19N but no change was observed in active Rac or Cdc42 upon transfection with Rac1T17N and Cdc42T17N, respectively. Since for both inhibition protocols cells are seeded on fibronectin, these results indicate that the dominant negative gene could inhibit RhoA even though the cells are plated on fibronectin, but the dominant negative gene could only reduce the activation of Rac and Cdc42 to background levels when the cells are plated on fibronectin. Consistent with our hypothesis of the involvement of RhoGT- Pases on non-viral gene transfer, inhibition of RhoGTPases led to a significant decrease in transgene expression. Although the exact mechanism of how RhoGTPases influence successful non-viral gene transfer cannot be determined from our data, we predict that the regulation of actin-microtubule dynamics, cell proliferation [15] and gene transcription via integrin-dependent intracellular signaling pathways [16] positively influence non-viral gene transfer. RhoGTPases have been implicated in endocytosis of bacteria and viruses by mammalian cells. For example, Rac1, RhoA, and Cdc42 are required for HeLa cell invasion by group B streptococcus [19], RhoA positively regulates the pathway by which Madin-Darby canine kidney (MDCK) cells internalize P. aeruginosa strain PA103 [18], and Rho C enhances macropino- cytosis and modulates entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors. Interestingly the effect on polyplex internalization was not the same for the bacterial toxins and transfection with dominant negative genes. While inhibition of RhoGTPases using TcdB and C3 transferase led to an increase in polyplex internalization, inhibition with dominant negative genes led to a decrease in internalization. We cannot offer a definitive reason for these findings. The observed increase in internalization on treatment with TcdB could be due to TcdB mediated activation of phospholipase C and D [34], leading to regulation of receptor mediated endocytosis [36,37]. The increase observed on C3 treatment correlates with our previous study where depolymer- ization of actin using cytochalasin D enhanced polyplex uptake in mMSCs cultured on fibronectin [6]. The observed decrease in internalization for inhibition with dominant negative genes could possibly be due to inhibition of macropinocytosis by dominant negative mutants of Cdc42 and Rac1, as was shown for bone marrow derived dendritic cells [38]. Since we found that exposure of mMSCs to fibronectin activates RhoGTPases (1.5- to 4-fold) and enhances transgene expression, we wanted to determine if further activation of RhoGTPases could further enhance transgene expression. RhoGTPases were activat- ed using calpeptin, EGF and transfection with constitutively active RhoGTPase mutants. Calpeptin (CP) is a cell permeable calpain inhibitor known to activate Rho leading to stress fiber formation [24,25]. Treatment with calpeptin and EGF did not influence the transgene expression or polyplex uptake. Although we show that PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35046