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. 2016 Feb 17;291(15):7902–7914. doi: 10.1074/jbc.M115.711564

FIGURE 2.

FIGURE 2.

TMR-r13 does not utilize the endocytic pathway to access the cytosolic space of cells. A, inhibitors of endocytic processes do not prevent cytosolic penetration. MCH58 cells were pretreated with 50 μm amiloride, 200 nm bafilomycin, or PBS supplemented with calcium and magnesium (vehicle) for 20 min. Cells were then incubated with inhibitors and with TMR-r13 (1 μm) for 1 h. Alternatively, cells were maintained at 4 °C during peptide incubation. The data represent the means of triplicate experiments and the corresponding standard deviations. NS (not significant) represents p > 0.05, and * represents p ≤ 0.05 compared with cells with no inhibitor at 37 °C. B, images of MCH58 cells treated with 5 μm TMR-r13 for the indicated times. Images are overlay of bright field, TMR-r13 (pseudocolored red), and SYTOX Blue (pseudocolored blue). A zoomed-in image shows distinct nucleolar staining of TMR-r13 without SYTOX Blue staining. Scale bar, 50 μm. C, TMR-r13 (5 μm, 1 h) fails to release DEAC-k5 from endocytic pathway when the endosomolytic agent dfTAT does. HDF cells were pre-incubated with DEAC-k5 (20 μm, 1 h, k is the one-letter code for d-lysine). Accumulation of DEAC-k5 (pseudocolored red) within endocytic organelles is established by co-localization with LysoTracker (pseudocolored green) (highlighted by arrows). Cells were then treated with TMR-r13 or dfTAT and imaged. Images are inverted monochromes of fluorescence emission and monochromes of bright field imaging. Scale bars, 50 μm. D, membrane translocation by TMR-r13 is not accompanied by penetration of the small molecule SYTOX Green or the peptide DEAC-K9. Cells were incubated for 1 h at 37 °C with both TMR-r13 (1 μm) and SYTOX Green (5 μm), a green cell-impermeable nuclear staining dye that can enter cells upon permeation of the plasma membrane. After peptide delivery, cells were washed and incubated with SYTOX Blue, a nuclear stain similar to SYTOX Green but with extinct excitation wavelength. TMR-r13 penetrated cells while excluding both SYTOX Green and SYTOX Blue. Similar results were obtained when SYTOX Green was substituted for DEAC-K9 (an oligolysine peptide labeled with the blue fluorophore DEAC). Fluorescence images are represented as inverted monochrome. Scale bars, 20 μm.