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Figure 2
(A) Identification of genes implicated in TAT-RasGAP317-326 internalization in Raji and SKW6.4 cells. The graphs depict the p-value (calculated using the MAGeCK procedure; see Materials and methods) for the difference in sgRNA expression between peptide-treated and control cells for the ~20,000 genes targeted by the CRISPR/Cas9 library. (B) Quantitation of TAT-RasGAP317-326 entry (top) and induced death (bottom) in wild-type (WT) and knock-out (KO) cells. The WT and the corresponding potassium channel KO versions of the indicated cell lines were pretreated or not for 30 min with 10 μM XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with or without 40 μM (Raji and SKW6.4 cells) or 80 μM (HeLa cells) TAT-RasGAP317-326. Internalization was recorded after 1 hr and cell death after 16 hr (Raji and SKW6.4) or 24 hr (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the WT versions of the different cell lines. (C) Quantitation of the modalities of TAT-RasGAP317-326 entry in WT and KCNQ5 KO Raji cells. Cells were incubated with FITC-TAT-RasGAP317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n = 165 cells for each time-point). The high percentage of cells with vesicular staining in the KO cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments.
Identification of potassium channels as mediators of direct translocation of cell-penetrating peptides (CPPs) into cells.