RhoGTPases and Non-Viral Gene Delivery Figure 1. RhoGTPase activation on fibronectin. (A) mMSCs were plated on uncoated or fibronectin (40 mg/mL) coated surfaces for 16 hours and cell morphology was studied by staining cells for actin using Alexa488 conjugated phalloidin (green), and for DNA using Hoechst 33258 dye (blue). Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 1006 magnification. (B) Stress fiber quantification. (C and D) Active RhoGTPase quantification was performed using GLISA specific for RhoA, Rac1,2,3 and Cdc42. Cells were culture in PDMS sheets and allowed to attach and spread before exposing them to protein modified plastic for 20 minutes or 1 hour. The average of three samples was plotted. The level of active RhoGTPase in samples exposed to fibronectin was compared with untreated sample using the unpaired t-test (two tail p value). The symbols * represents a significant change to the level of p,0.05. The error bars represent the standard deviation in all plots. doi:10.1371/journal.pone.0035046.g001 active mutants of either RhoA (RhoA-Q63L), or Rac1 (Rac1- Q61L) or Cdc42 (Cdc42-Q61L) [23]. At least 80% cell viability was maintained in cells expressing EGFP control plasmid and active genes 16 hours post plating cells on fibronectin (at time of bolus transfection) and 48 hours post gene delivery (Fig. S2D). Transfection with constitutively active RhoA mutant resulted in an increase in stress fiber formation (Fig. 5A, B). Further, a significant increase in the amount of active RhoA, Rac1 and Cdc42 was observed for cells transfected with the constitutively active genes (p at least,0.05, Fig. 5D). Similar to the results obtained for CP and EGF further activation of RhoGTPases did not enhance transgene expression further over that observed for cells seeded on fibronectin and transfected with EGFP (Fig. 5D). Expression of constitutively active form of RhoA, Rac1 and Cdc42 did not affect internalization (Fig. 5E). These results confirm that further activation of RhoA, Rac1 or Cdc42 over what fibronectin achieves on its own (Fig. 1 C, D) does not alter transgene expression. Activation of RhoGTPases for cells plated on collagen I enhances transgene expression Activation of RhoGTPases for cells plated on collagen I was significantly lower compared to cells plated on fibronectin (Fig. 6 vs. Fig. 1). Thus, we used transfection experiments on collagen I to further test our hypothesis of the importance of active RhoGT- Pases on non-viral gene transfer. Rho was enhanced 4-fold on fibronectin coated surfaces but only 1.5-fold for cells plated on collagen I. Rac was enhanced 1.5-fold for cells plated on fibronectin but inhibited by 50% for cells plated on collagen I. Cdc42 was enhanced by 2-fold for cells plated on fibronectin, but inhibited by 50% for cells seeded on collagen I (Fig. 6A). Cells were transfected with constitutively active RhoA, Rac1 and Cdc42 and replated on collagen I coated surfaces for 16 hours before transfection. Rac1 and Cdc42 activation were able to enhance the transgene expression by 4.1 and 6.7 fold compared to EGFP mock transfected cells respectively, while RhoA activation had no difference (Fig. 6B). Since RhoA was activated by cell exposure to collagen I (Fig. 6A), this result shows that further activation of RhoA is not beneficial to achieve higher transgene expression similarly to what was observed for cells plated on fibronectin. Rho effector protein Rho-Kinase (ROCK) and PKC mediated phosphorylation modulate gene transfer ROCK was inhibited using Y27632 (Y2) and PKC was inhibited using staurosporine (ST). Treatment with Y27632 reduced the number of actin stress fibers while treatment with PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35046