dence that matrix stiffness regulates intracellular uptake of MK2-inhibitor peptides. Not only is there an increase in uptake as observed by flow cytometry, but there is a functional difference as characterized by the production of proinflammatory cytokines. Based on the quantification of surface fibronectin protein, which showed equivalent amounts of ECM GSK-2256294 protein between the soft and stiff polyacrylamide gel, the difference in YARA uptake can be attributed directly to the effect of stiffness. Characterization of the fibronectin protein is important because several investigators have demonstrated that cells respond morphologically to varying amounts of matrix proteins. We wanted to ensure that the cell response observed could be appropriately attributed to matrix stiffness and not the matrix proteins. Importantly, the mechanism of uptake of FITC-YARA does not appear to change when cells are seeded on soft substrates. Mesothelial cells seeded on tissue culture plastic show inhibition of uptake at 4uC and with the removal of cholesterol using the pharmacological inhibitor, MbCD. Figure 7 demonstrated that FITC-YARA uptake on soft substrates was also energy-and cholesterol-dependent. MK2 is known to regulate the expression of several proinflammatory cytokines, however we focused on the down regulation of TNF-a protein expression as a marker of YARA activity. In previous studies we have noted that cytokine expression is down regulated at 6 hours and continues to be down regulated at 24 hours with the difference between expression in control and treated cells maximized at the later time point. We believe that the MCE Company 755038-02-9 prolonged ability of the peptide to suppress cytokine protein expression is due to uptake into and prolonged residence time in caveolae. This is consistent with work by Kim, et al. where they saw prolonged retention of TAT-M13-phage in caveolae, and is subject to further investigation. The concentration required to demonstrate efficacy in this cell line was equiv
