Fig. 1. Structure and cellular uptake of PP75.

(A) Chemical structure of PP75 where the ratio between carboxylate and phenylalanine on the side chain R is 37:63. (B) FACS analysis was used to quantitate the relative uptake of fluorescently labeled PP75 (PP75-AF647) by U251 cells. Cells were treated with PP75-AF647 alone at 37°C or at 4°C. Cells were also treated with PP75-AF647 in combination with sucrose, a clathrin-mediated endocytosis inhibitor, or nystatin, a caveolin-mediated endocytosis inhibitor (both at 37°C). The mean fluorescence intensity values were normalized to that of cells treated with PP75-AF647 alone at 37°C. Error bars represent the standard deviation of triplicate experiments. The results indicate that PP75-AF647 is predominantly taken up by cells via clathrin-mediated endocytosis. (C) Cells were incubated with PP75-AF648 (red fluorescence) and either transferrin-AF488 (green fluorescence), a clathrin endocytosis marker, or BODIPY-LacCer (green fluorescence), a caveolin endocytosis marker, and visualized using confocal fluorescence microscopy. Overlay of the red and green channels reveals areas of co-localization (yellow fluorescence) and quantitation of this co-localization was performed using MetaMorph software. The mean percentage overlap of PP75-AF647 with transferrin-AF488 or BODIPY-LacCer is shown to the right of the microscopy images and error bars represent the standard deviation of triplicate experiments. Scale bars in the photos represent 20 μm. The greater overlap of PP75-AF647 with transferrin-AF488 supports the preferential uptake of PP75 via clathrin-mediated endocytosis compared to caveolin-mediated endocytosis.