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Figure 1
(A) Depiction of the different modes of cell-penetrating peptide (CPP) entry into cells. Confocal microscopy was performed on the indicated cell lines incubated for 1 hr with 40 μM FITC-TAT-RasGAP317-326 in RPMI, 10% fetal bovine serum (FBS). Cells were washed with PBS prior to visualization. Vesicular staining is indicative of CPP endocytosis while diffuse cytosolic staining is a consequence of CPP direct translocation into cells. Scale bar: 10 μm. (B) Quantitation of the different modes of CPP entry as a function of time (FITC-TAT-RasGAP317-326 continually present in the media) using the experimental conditions presented in panel A. Types of staining were visually quantitated as indicated in Figure 1—figure supplement 1A (n = 157 cells per condition). There was no indication of fluorescence quenching, due to endosomal acidification, preventing the detection of CPP-containing endosomes (in at least during the first hour of CPP exposure) (Figure 1—figure supplement 1B). TAT-RasGAP317-326 enters cells via endocytosis and direct translocation, but only direct translocation mediates its biological activity and leads to cell death (Figure 1—figure supplement 4). Results correspond to the average of three independent experiments.
TAT-RaGAP317-326 cellular entry modes.