Supporting Figures

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Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Fig. 7. Chemical structures of the cargo molecules used in our study. (a) Dansyl dye. (b) Sequence of PNA. (c) Sequence of ODN. (d) Phalloidin.

Fig. 8. Fluorescence and bright-field confocal images of JC breast adenocarcinoma cells incubated (for 1 h) with the noncleavable pHLIP-Ph-TRITC construct (1 mM) at pH 6.5 and washed with buffer at pH 6.5 (Right). Cells were washed with buffer at pH 7.4, and the pHLIP-Ph-TRITC construct was removed, showing that penetration is reversible.

Fig. 9. Cell toxicity and membrane leakage tests. (a) Incubation of HeLa cells with pHLIP at concentrations up to 16 mM for 24 h under physiological conditions did not affect cell viability. Untreated cells were taken as 100%. (b) Membrane leakage was tested by incubation of HeLa cells at various concentrations of peptide (up to 10 mM) at pH 7.4 and 6.5 for 1 h followed by addition of cell-impermeable agents, nuclear-staining SYTOX-Orange (0.5 mM) (light columns), and Ph-TRITC (2 mM) (dark columns) for 10 min in the presence of peptide and then washed with PBS buffer (pH 7.4). The cell membrane was subsequently disrupted by adding 0.5% Triton X-100 and a new portion of the 2 mM Ph-TRITC and 0.5 mM SYTOX-Orange followed by washing with PBS. The fluorescence values obtained in the absence or presence of various concentrations of peptide were all of the same and ~10% of that obtained after cell membrane disruption by Triton X-100.

Fig. 10. Fluorescence and phase-contrast images of the same HeLa cells were taken before (Left) and 5 min after adding dissociation solution (Right). Weakly stained cells became round (arrows), and strongly stained cells remained unchanged, showing the stabilization of the cytoskeleton by Ph-TRITC delivered by pHLIP.