Article Title: Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore
Figure Lengend Snippet: Potassium channels maintain plasma membrane polarization that is required for CPP entry into cells a , Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or TRAM-34. The grey and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. b , Effect of cellular depolarization (left of the grey zone) and hyperpolarization (right of the grey zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP 317-326 for one hour. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. c , Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n > 150 cells) incubated for one hour with 10 μM FITC-CPP in depolarizing (2 μg/ml gramicidin) or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (see Supplementary Fig. 9g ). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. The confocal images were acquired in the middle of the cells based on Hoechst fluorescence. d , Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin blue lines.
Article Snippet: XE-991 and TRAM-34 (Alomone labs, ref. no. X-100 and T-105 respectively) was dissolved in DMSO at 100 mM and stored at -20 °C.
Techniques: Knock-Out, Expressing, Flow Cytometry, Quantitation Assay, Incubation, Confocal Microscopy, Fluorescence