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    Alomone Labs tram 34
    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 <t>TRAM-34)</t> 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.
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    Images

    1) Product Images from "Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore"

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

    Journal: eLife

    doi: 10.7554/eLife.69832

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    2) Product Images from "Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells"

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

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

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.
    Figure Legend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

    Techniques Used: Translocation Assay, Expressing, CRISPR, Quantitation Assay, Knock-Out, Incubation, Staining

    Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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.

    Techniques Used: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.
    Figure Legend 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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Techniques Used: Knock-Out, Expressing, Quantitation Assay, Flow Cytometry, Two Tailed Test

    3) Product Images from "Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore"

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

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    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.
    Figure Legend 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.

    Techniques Used: Knock-Out, Expressing, Flow Cytometry, Quantitation Assay, Incubation, Confocal Microscopy, Fluorescence

    Identification of potassium channels as mediators of direct translocation of CPPs into cells a , Identification of genes implicated in TAT-RasGAP 317-326 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-RasGAP 317-326 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 minutes 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-RasGAP 317-326 . Internalization was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP 317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the wild-type versions of the different cell lines. c , Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The high percentage of cells with vesicular staining in the knock-out (KO) cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments.
    Figure Legend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells a , Identification of genes implicated in TAT-RasGAP 317-326 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-RasGAP 317-326 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 minutes 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-RasGAP 317-326 . Internalization was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP 317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the wild-type versions of the different cell lines. c , Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The high percentage of cells with vesicular staining in the knock-out (KO) cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments.

    Techniques Used: Translocation Assay, Expressing, CRISPR, Quantitation Assay, Knock-Out, Incubation, Staining

    4) Product Images from "Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells"

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

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

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.
    Figure Legend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

    Techniques Used: Translocation Assay, Expressing, CRISPR, Quantitation Assay, Knock-Out, Incubation, Staining

    Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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.

    Techniques Used: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.
    Figure Legend 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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Techniques Used: Knock-Out, Expressing, Quantitation Assay, Flow Cytometry, Two Tailed Test

    5) Product Images from "The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells"

    Article Title: The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells

    Journal: Vascular pharmacology

    doi: 10.1016/j.vph.2018.01.004

    NAGly and abn-cbd induce BK ca -dependent endothelial cell hyperpolarization in mouse aorta independently of gap junctions and GPR18. (A) A lack of effect of phenylephrine (1 μM) on the membrane potential of endothelial cells in isolated mouse aorta. Note that subsequent administration of Ach produces a hyperpolarization. (B) In the endothelium of isolated rat aorta, phenylephrine produces depolarization with membrane potential oscillations. (C) Effect of 10 μM NAGly (n = 5) on endothelial membrane potential in isolated mouse aorta. (D) Effect of 30 μM NAGly on endothelial membrane potential in isolated mouse aorta (n = 4). (E) Membrane potential recording showing the effect of paxilline (1 μM) on the hyperpolarization induced by NAGly (n = 3). (F) Membrane potential recording showing a failure of TRAM-34 to suppress the hyperpolarization to 10 μM abn-cbd in excised mouse aorta. (G) Intracellular perfusion with GPR18 antibody (1:500) fails to prevent endothelial cell hyperpolarization to 10 μM NAGly in isolated mouse aorta (n = 4). Recordings were commenced in the presence of 100 μM glycyrrhetinic acid, a gap junction inhibitor. (H) Bars showing the magnitude of shifts in endothelial membrane potential in isolated mice aorta induced by NAGly (10 μM and 30 μM) alone and combination of 10 μM NAGly and paxilline (1 μM) and GPR18 antibody. (I) Effect of NS1619 on the membrane potential of endothelial cells in excised mouse aorta. (J) Bars showing mean values of endothelial cell hyperpolarization to 10 μM (n = 6) and 30 μM NS1619 (n = 5).
    Figure Legend Snippet: NAGly and abn-cbd induce BK ca -dependent endothelial cell hyperpolarization in mouse aorta independently of gap junctions and GPR18. (A) A lack of effect of phenylephrine (1 μM) on the membrane potential of endothelial cells in isolated mouse aorta. Note that subsequent administration of Ach produces a hyperpolarization. (B) In the endothelium of isolated rat aorta, phenylephrine produces depolarization with membrane potential oscillations. (C) Effect of 10 μM NAGly (n = 5) on endothelial membrane potential in isolated mouse aorta. (D) Effect of 30 μM NAGly on endothelial membrane potential in isolated mouse aorta (n = 4). (E) Membrane potential recording showing the effect of paxilline (1 μM) on the hyperpolarization induced by NAGly (n = 3). (F) Membrane potential recording showing a failure of TRAM-34 to suppress the hyperpolarization to 10 μM abn-cbd in excised mouse aorta. (G) Intracellular perfusion with GPR18 antibody (1:500) fails to prevent endothelial cell hyperpolarization to 10 μM NAGly in isolated mouse aorta (n = 4). Recordings were commenced in the presence of 100 μM glycyrrhetinic acid, a gap junction inhibitor. (H) Bars showing the magnitude of shifts in endothelial membrane potential in isolated mice aorta induced by NAGly (10 μM and 30 μM) alone and combination of 10 μM NAGly and paxilline (1 μM) and GPR18 antibody. (I) Effect of NS1619 on the membrane potential of endothelial cells in excised mouse aorta. (J) Bars showing mean values of endothelial cell hyperpolarization to 10 μM (n = 6) and 30 μM NS1619 (n = 5).

    Techniques Used: Isolation, Mouse Assay

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    Alomone Labs tram34
    Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and <t>apamin+tram34</t> (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P
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    Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P

    Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

    Techniques: Isolation, Mass Spectrometry, Activity Assay

    Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels. A : (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B : Representative force myograph trace obtained from an ovx artery. C and D : Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their ( C ) change in tone and ( D ) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C : Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D : Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI 2 ) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels. A : (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B : Representative force myograph trace obtained from an ovx artery. C and D : Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their ( C ) change in tone and ( D ) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C : Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D : Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI 2 ) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

    Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

    Techniques: Blocking Assay

    IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels. A : Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B : Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels. A : Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B : Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P

    Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

    Techniques: Activity Assay, Mouse Assay

    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.

    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

    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.

    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

    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour 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

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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-RasGAP 317-326 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 minutes with XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. (C) Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three 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, Expressing, CRISPR, Quantitation Assay, Knock-Out, Incubation, Staining

    Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake 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 condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 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

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. 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, Quantitation Assay, Flow Cytometry, Two Tailed Test