PKC and ROCK inhibition, inhibit gene transfer in cells plated on fibronectin.

<p>mMSCs were cultured for 16 hours on fibronectin coated plates prior to treatment with 10 µM Y27632 (Y2) or 100 nM Staurosporine (ST) for 2 hours to inhibit ROCK and PKC, respectively. (<b>A</b>) 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 40× magnification. Immediately after treatment with inhibitor, the medium was replaced and bolus transfection was done. (<b>B</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 (<b>C</b>). 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.