Figure 2.

Analysis of peptide internalization by confocal fluorescence microscopy. Mammalian breast epithelial cancer cells (MDA-MB-453) were grown to 95% confluence in a 24-well glass-bottom plate and incubated with 10 μM fluorescently-labeled peptide in PBS for 1 hour at 37°C or 4°C.⁸ Low-temperature conditions were used to demonstrate the extent to which the uptake process was energy-dependent. Alternatively, cells were pre-treated with 80 μM dynasore (a dynamin inhibitor that prevents clathrin-mediated endocytosis)²⁰ for 30 min at 37° C prior to incubation with dye-labeled peptides at the same temperature. Cells were washed thoroughly in PBS prior to imaging by confocal microscopy. While peptides were incubated in PBS, we also verified their stability in buffered human serum as described previously (Supplementary Figure S3).¹²