Activated Rho, Rac and Cdc42 were confirmed by performing respective activation G-LISA assays (RhoA, Rac1,2,3 and Cdc42 G-LISA Activation Assays; Cytoskeleton) with lysates from activated and control samples as per manufacturer’s protocol. Overnight serum starved cells were treated as the control sample. To assess activity, samples were subsequently treated with 25 mg/ ml calpeptin for 10 minutes or with 50 ng/ml EGF for 2 minutes. Activity of RhoA, Rac and Cdc42 respectively was assessed using a GLISA assay and normalized to values obtained for control lysates. Transient inhibition and activation of RhoGTPase Specific inhibition and activation of the RhoGTPases was done by a method described by Burnham et al. [19] with minor modification. Plasmids expressing dominant negative Rac1 (pcDNA3-EGFP-Rac1-T17N), RhoA (pcDNA-EGFP-RhoA- T19N) and Cdc42 (pcDNA-EGFP-Cdc42-T17N), as well as plasmids expressing constitutively active mutants of RhoA (pcDNA-EGFP-RhoA-Q63L), Rac1 (pcDNA-EGFP-RhoA- Q61L) and Cdc42 (pcDNA-EGFP-Cdc42-Q61L) were kindly provided by Dr Garry Bokoch (The Scripps Research Institute, La Jolla, CA) [23,52]. 56104 and 256104 cells were plated per well in a 24 and 6 well plate respectively, and cultured for 24 hours prior to transfection. For every 1 mg of DNA, 2 ml of LipofectamineTM 2000 transfection reagent was added to the DNA solution, vortexed for 15 seconds and incubated at room temperature for 20 minutes. Complexes were added directly to the medium of the plated cells at a final DNA concentration of 1 mg per well for 24- well plates and 2.5 mg per well for 6-well plates. Analysis of GFP expression in transfected cells using FACS showed 30–40% cells to be successfully transfected with dominant negative or constitutively active genes and control plasmid (Fig. S2 A, B). 24 hours post transfection, cells were harvested and used to study the effect of RhoGTPase inhibition or activation on transgene expression as well as internalization. Activation or inhibition was analyzed by assessing RhoGTPases activity using respective GLISA assays (RhoA, Rac and Cdc42 G-LISA Activation Assays; Cytoskeleton) as per manufacturer’s protocol. Cells transfected with pEGFP using LipofectamineTM 2000, were used as control for studies involving transfection with dominant negative and constitutively active genes. For assessing activation, 24 hours post transfection with constitutive active genes, cells were serum starved overnight and then harvested for performing respective activation G-LISA assays and studying morphology. For assessing inhibition, 24 hours post transfection with dominant negative genes, cells were replated on fibronectin coated surface overnight and harvested for performing GLISA assays and studying morphology. Transfection pEGFP-luc plasmid was purchased from clontech (Palo Alto, CA) and expanded using a Giga Prep Kit from Qiagen following the manufacturer’s protocol. Linear poly-(ethylene imine) (25 kDa, PEI) was purchased from Polysciences (Warrington, PA). The cells were allowed to attach and cultured under various conditions as per required for the experiments mentioned. For gene transfer studies, DNA/PEI polyplexes were formed by mixing equal volumes of plasmid DNA with PEI. For every 1 mg of DNA, 1.65 mg of PEI was added to the DNA solution to get N/P of 12, vortexed for 15 seconds and incubated at room temperature for 15 minutes. Polyplexes were added directly to the medium of the plated cells at a final DNA concentration of 0.5 mg for 48-well plates. Salt was added directly to the wells post addition of transfection solution to get a final concentration of 150 mM NaCl. Transfection was quantified at 48 hours post transfection using the RhoGTPases and Non-Viral Gene Delivery Promega Luciferase Assay System following the manufacturer’s instructions. Subsequently, the total amount of protein in samples was analyzed using Peirce BCA assay with or without compat-Able protein assay preparation kit as per manufacturer’s protocol. Experiments were carried out in triplicates and results were expressed as relative light units (RLU) per mg of cell protein. Internalization of polyplexes Plasmid DNA (pEGFPluc) and the fluorescent DNA-interca- lator YOYO-1 or YOYO-3 were mixed at a ratio of 1 dye molecule per 50 base pairs and allowed to complex for 60 minutes at room temperature. YOYO-1 or YOYO-3 labeled DNA was then used to prepare PEI/DNA complexes at a N/P of 12, bolus transfection was performed and cells were exposed to polyplexes for 2 hours. For YOYO-1 labeled complexes, cells were washed with PBS, trysinized with 50 ml of 0.25% trypsin-EDTA and suspended in 350 ml of 0.04% tryphan blue in 1% BGS in PBS to quench the flourescence of extracellular associated DNA. For YOYO-3 labeled complexes, cells were washed twice for 10 minutes with cellscrub to remove extracellular associated DNA, trysinized and suspended in 1% BGS in PBS. YOYO-1 fluorescent cells having were detected by flow cytometry with a FACScan X and data was analyzed with CELLQuest (Beckton Dickinson). YOYO-3 fluorescent cells were detected by flow cytometry using BD LSR II and data was analyzed with FACSDiVa (Beckton Dickinson). Experiments were performed in triplicates analyzing 7000 or 10000 total events per sample. Microscopy and quantification of stress fibers D1 cells were plated on fibronectin coated plastic coverslips prior to culture. Immediately after treatment with Rho protein activators or inhibitors, cells were fixed with 5% paraformalde- hyde for 15 minutes and permeabilized with 0.1% tritonX100 in 16 PBS for 3 minutes. The cells were stained for actin using Alexa488-phalloidin and for DNA using Hoechst 33258 dye. The staining solution was added in each well and left in dark for 30– 60 minutes at room temperature followed by washings with 0.05% tween-20. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 1006 magnification. The number of stress fibers per cell was quantified by counting the individual fibers observed in 1006 pictures taken of cells in the respective condition. Statistical methods All statistical analyses were performed using the computer program Instat (GraphPad, San Diego, CA). Experiments were statistically analyzed using a one-way ANOVA followed by a post- hoc test if the ANOVA result was p,0.05. Experiments were statistically analyzed using the Tukey test, which compares all pairs of columns, using a 95% confidence interval or using the Dunnet test which compares all columns versus a control column or using the unpaired t-test (two tail p value), which compares two different columns. Statistical significance was determined using a 95% confidence interval. Data is plotted as the mean of at least three independent measurements and standard deviation. Supporting Information Figure S1 Effect of inhibitors and activators on cell viability. To study the effect of RhoGTPase inhibition on gene transfer, mMSCs were plated on fibronectin (40 mg/mL) coated tissue culture plastic surfaces for 16 hours prior to being treated with 0–300 ng/ml TcdB for 4 hours in serum free media, or 0– 1 mg/ml C3 transferase for 4 hours. Cells were transfected PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35046