ws12  (Alomone Labs)

Journal: eLife

Article Title: Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

doi: 10.7554/eLife.69832

Figure Lengend Snippet: Potassium channels regulate the cellular internalization of various TAT-bound cargos. ( A ) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type (WT) condition using ANOVA multiple comparison analysis with Dunnett’s correction. ( B ) Representative microscopy images of WT and KCNQ5 knock-out (KO) Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 μM TAT-Cre for 48 hr. The results correspond to one of three independent experiments. ( C ) Internalization, recorded by flow cytometry, of FITC-D-JNKI1 after 1 hr of incubation in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.

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: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing, Flow Cytometry, Incubation

Journal: eLife

Article Title: Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

doi: 10.7554/eLife.69832

Figure Lengend Snippet: Potassium channels modulate direct cell-penetrating peptide (CPP) translocation, but not endocytosis. ( A ) Same as Figure 2C , but for wild-type (WT), KCNN4 and KCNK5 SKW6.4 knock-out (KO) cells. The results correspond to the average of three independent experiments. ( B ) As panel A, but for WT and KCNN4 KO HeLa cells. The results correspond to the average of three independent experiments. ( C ) Quantitation by flow cytometry of 20 μg/ml AlexaFluor488-transferrin (left) or 200 μg/ml 10 kDa FITC-Dextran (right) internalization in the indicated WT cell lines and their corresponding KO versions, pretreated or not for 30 min with the XE-991 (10 μM) or TRAM-34 (10 μM) potassium channel inhibitors. Transferrin and dextran internalization was allowed to proceed for 60 min (still in the presence of inhibitors when these were used in the 30 min pre-incubation period). To quench membrane-bound fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis. The independent experiment replicates are color-coded. ( D ) Assessment of FITC-TAT-RasGAP 317-326 cell surface binding on WT and KCNQ5 KO Raji cells after 60 s of incubation (top), as well as associated peptide internalization after 1 hr of treatment (bottom). The results correspond to at least five independent experiments.

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: Translocation Assay, Knock-Out, Quantitation Assay, Flow Cytometry, Incubation, Fluorescence, Binding Assay