Figure 3. RhoGTPase inhibition using dominat negative gene products decreases transgene expression. mMSCs were transient- ly transfected with dominant negative forms of RhoA, Rac1 or Cdc42 with a plasmid also containing EGFP. 24 hours post transfection, cells were replated on fibronectin coated plates and cultured for 16 hours prior to bolus transfection. Cells transfected with pEGFP were used as control. (A) Alexa488 conjugated phalloidin (green), and Hoechst dye (blue) staining. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 1006 magnification. (B) Stress fiber quantifica- tion. Statistical analysis was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p value was 0.0015. The symbol * represents a significant change in stress fibers with respect to untreated cells to the level of p,0.05. (C) Active Rho, Rac and Cdc42, was assessed for cells replated on fibronectin for 16 hours using GLISA assays specific for RhoA, Rac1,2,3 and Cdc42. Statistical analysis was done using the unpaired t-test (two tail p value). The symbol * represents a significant change to the level of p,0.05. (D) 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 using YOYO-3 labeled polyplexes 2 hours post transfection using flow cytometry. 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 gene transfer and internalization was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p values were 0.0219 and ,0.0001 for transfection (D) and internalization (E) respectively. The symbols * and ** represents a significant change with respect to cells transfected with control plasmid (pEGFP) to the level of p,0.05 and p,0.01, respectively. doi:10.1371/journal.pone.0035046.g003 RhoGTPases and Non-Viral Gene Delivery Figure 4. Effect of RhoGTPase activation using calpeptin (CP) and epidermal growth factor (EGF) on gene transfer. mMSCs cells were cultured for 8 hours on fibronectin wells, 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. (A) After treatment, the changes in cell morphology were visualized through lexa488 conjugated phalloidin (green) and for DNA using Hoechst 33258 dye (blue) staining. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 1006 magnification. (B) Stress fiber quantification. (C) Active RhoGTPase quantification after treatment was performed using GLISA specific for RhoA, Rac1,2,3 and Cdc42. (D) Transfection was performed immediately post treatment in serum free media. 4 hours post transfection media was replaced with complete media. 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 of YOYO-1 labeled polyplexes. A total of 7000 events were analyzed per sample. Statistical analysis for the level of active RhoGTPase, transgene expression and internalization, was done using the unpaired t-test (two tail p value) where treated sample was compared with untreated sample. The symbols ** and *** represents a significant change to the level of p,0.01 and p,0.001, respectively. doi:10.1371/journal.pone.0035046.g004 in inhibited gene transfer compared to cells seeded on uncoated surfaces [6]. Here we aimed to determine if RhoGTPase activation was one of the reasons for the observed enhancement in transgene expression for cells plated on fibronectin-coated surfaces. RhoGTPases have been primarily implicated as the controlling factors in guiding cell motility, morphology, contractility and proliferation [17]. RhoGTPases have also been implicated in regulating phagocytosis [28], endocytic trafficking [29], as well as mediating the various steps of vesicular trafficking [30]. Thus, RhoGTPases can play a role in polyplex uptake and polyplex intracellular trafficking. Fibronectin has been previously shown to enhance signaling through RhoGTPases [11,14,31,32]. Initial activation of Rac and Cdc42 by fibronectin has been shown to PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35046