Figure 7. PKC and ROCK inhibition, inhibit gene transfer in cells plated on fibronectin. mMSCs were cultured for 16 hours on fibronectin coated plates prior to treatment with 10 mM Y27632 (Y2) or 100 nM Staurosporine (ST) for 2 hours to inhibit ROCK and PKC, respectively. (A) Changes in mMSC cell morphology were visualized through staining for actin using Alexa488 conjugated phalloidin and for DNA using Hoechst 33258 dye. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 406 magnification. Immedi- ately after treatment with inhibitor, the medium was replaced and bolus transfection was done. (B) Transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. Internalization was assessed 2 hours post transfection using flow cytometry and YOYO-1 labeled polyplexes (C). A total of 7000 events were analyzed per sample. Statistical analysis was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p value was 0.0003 and ,0.0001 for transgene expression and internalization, respectively. The symbols ** represents a significant change to the level of p,0.01. doi:10.1371/journal.pone.0035046.g007 (Sigma-Aldrich, St. Louis, MO) containing 10% bovine growth serum (BGS, Hyclone, Logan, Utah) and 1% penicillin/strepto- mycin antibiotics (Invitrogen, Grand Island, NY) and cultured at 37uC and 5% CO2. Protein coating Stock solution fibronectin (1 mg/ml, Millipore, Billerica, MA) was diluted to 40 mg/ml in phosphate buffered saline (PBS), and added in each well of a tissue culture plate. The plate was incubated over night at 4uC in a humid environment, followed by incubation at 37uC for 2 hours [48]. The solution was removed and wells were washed twice with PBS to remove unbound proteins. Wells were further incubated with BSA (1% in PBS) for 30 minutes at 37uC followed by two washings with PBS. Analysis of RhoGTPase activation by fibronectin or collagen I Poly-dimethoxy silane (PDMS) sheets were made by mixing Sylgard 184 PDMS prepolymer thoroughly in a 10:1 mass ratio of silicone elastomer to curing agent, degassing and casting in a square dish at 60uC overnight. After the cured PDMS sheets were RhoGTPases and Non-Viral Gene Delivery removed, they were cut into strips and oxygen plasma treated using Technics RIE (Reactive Ion Etching) for 3 minutes at 100 mTorr of O2, 100 W or 15 minutes at 200 mTorr of O2, 200 W. The PDMS strips were subsequently placed in a 1:1 v/v methanol and hydrochloric acid solution for 30 minutes, then dried and incubated overnight in a 5% v/v amino-propyl triethoxysilane (APTES, Sigma-Aldrich, St. Louis, MO) solution in ethanol, at room temperature and in inert atmosphere [49]. The sheets were sterilized in 70% ethanol prior to cell plating. The cells were cultured for a day till confluent and then serum starved overnight before exposing them to fibronectin or collagen I coated surfaces. Five minutes before exposing cells to fibronectin or collagen I, sodium orthovanadate (0.1 mM) was added to the cell culture media. Sodium orthovanadate is an inhibitor of protein tyrosine phosphatases and alkaline phosphatases and was used to preserve the phosphorylation of RhoGTPases through inhibiting endogenous phosphatases [14]. The PDMS cell sheets were exposed to fibronectin or collagen I for 20 min or 1 hour, which have been shown to be the duration of RhoGTPase activation in different cells after interaction with the extracellular matrix [11,14,31,32]. After exposure to ECM protein at 37uC, the strips were placed in Petri dishes on ice and rinsed once with ice cold PBS supplemented with 0.2 mM sodium orthovanadate. The lysis buffer provided with the GLISA kit (Cytoskeleton, Inc., Denver, CO) was modified to include 5 mM sodium orthovanadate, 50 mM sodium fluoride and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), along with 16 protease inhibitor cocktail provided with Kit [14,50] After aspirating all liquid from the PDMS strips, 100 ul of lysis buffer was added and scraped. Cell lysate was immediately clarified by centrifugation at 9,000 rcf, 4uC for 2 minutes, aliquoted into microcentrifuge tubes and snap frozen in liquid nitrogen, as indicated by the manufacture’s protocol. After 1 or 2 days, the frozen aliquots were analyzed and RhoGTPases activity assessed, as per manufacture’s protocol. Chemical inhibition of RhoGTPases Difficile toxin B (TcdB, List Biologicals, Campbell, CA) was used to pharmacologically inhibit Rho, Rac and Cdc42 [18– 20,28,51]. Rho was specifically inhibited using cell permeable C3 transferase (C3, Cytoskeleton, Denver, CO) [18,28,51]. mMSCs were seeded on fibronectin coated 48-well plates or 24-well plates at cell densities of 20,000 and 50,000 cells/well respectively. The cells were allowed to attach for 14–15 hours before the media in the wells was replaced with serum free media containing 0–1 mg/ ml C3 transferase (C3) or 0–300 ng/ml TcdB for 4 hours. Immediately post treatment, media was changed with serum containing media and DNA/PEI internalization and overall transgene expression studied. To assess RhoGTPase inhibition, cell morphology was analyzed and respective GLISA assays were performed, immediately after inhibitor treatment. Chemical activation of RhoGTPase Cells were allowed to attach on fibronectin-coated plates for 8 hours followed by overnight incubation in serum free media. Rho was pharmacologically activated by s incubating cells in presence 25 mg/ml Calpeptin (CP, Cytoskeleton, Inc., Denver, CO) for 10 minutes while Rac and CDC42 were activated by incubating cells in presence of 50 ng/ml Epidermal growth factor (EGF, Cytoskeleton) for 2 minutes. Immediately after treatment cell morphology, internalization and gene expression were analyzed. For analysis of non-viral gene transfer, cells were transfected with polyplexes in serum free media for 2–4 hrs, following which media was replaced with serum containing media. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35046