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 lipofectamine™2000. 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.