Figure 3
The free tetrameric peptides bind to and mediate internalization.
(A). Cells were incubated with 10 nM of the SAQdot605 conjugated to the tetrameric peptide via a streptavidin-biotin interaction. Images were obtained on a Leica DM5500 Fluorescence Microscope at 400× total magnification. The scale bar represents 20 microns. The top panels are H2009 cells incubated with the indicated peptides while the bottom panels are H1299 cells. The red color represents the peptide-Qdot conjugates; blue is DAPI staining of the nuclei; the green is the cell surface stained with wheat germ agglutinin. The peptides do not bind the non-targeted cell line. In all cases, the micrographs for each peptide/cell pair are obtained at the same gain. (B). H460.1-SAQdot605 was incubated with H2009 cells (left panel) and MCF7 cells (right panel). MCF7 cells have a high selectivity for the peptide and are more suitable for microscopy experiments than H460 cells due to their larger size and higher cytoplasm/nucleus ratio. The target cells have localized patches of peptide binding that are excluded from cell-cell contacts as indicated by white arrows. Hazy staining is seen in the extracellular space for the MCF7 cells (indicated by yellow arrows) but not the H2009 cells signifying binding to shed or secreted proteins.
