4 aminopyridine  (Alomone Labs)


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    Structured Review

    Alomone Labs 4 aminopyridine
    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of <t>4-aminopyridine</t> (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p
    4 Aminopyridine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells"

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006168

    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p
    Figure Legend Snippet: Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p

    Techniques Used: Inhibition, BrdU Incorporation Assay

    Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p
    Figure Legend Snippet: Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p

    Techniques Used: Inhibition, MTT Assay

    Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .
    Figure Legend Snippet: Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .

    Techniques Used: Inhibition, Mass Spectrometry

    Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.
    Figure Legend Snippet: Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.

    Techniques Used: Patch Clamp, Activation Assay, Mass Spectrometry

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    Alomone Labs ns8593
    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with <t>NS8593</t> (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p
    Ns8593, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Alomone Labs ns1643
    <t>NS1643</t> inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p
    Ns1643, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Alomone Labs 4 aminopyridine
    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of <t>4-aminopyridine</t> (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p
    4 Aminopyridine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 aminopyridine/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    4 aminopyridine - by Bioz Stars, 2022-08
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    Image Search Results


    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Incubation, Flow Cytometry

    TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, Mouse Assay, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Plex Assay, Derivative Assay, Mouse Assay, Incubation

    NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits expression of N-cadherin while augmenting E-cadherin ( A ) Relative expression level of mRNA encoding for N-Cadherin or ( B ) E-cadherin by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits migration. ( A ) Western blot analysis showing expression of Kv11.1 in HT29, SW480, and FET colon cancer cell lines. For the original Western blots, see Supplementary Figure S1 . ( B ) Representative Kv11.1 current in control HT29 cells in the presence of DMSO (control), NS1643 (activator; 50 µM) alone, or NS1643 + E4031 (blocker; 5 µM), with the voltage protocol shown. ( C ) Representative diary plot depicting the Kv11.1 peak currents (at −100 mV) as a function of time in HT29 cells before and after application of NS1643 alone or in combination with E4031. ( D ) Wound-healing assay with HT29, SW480, or FET cells treated with DMSO or 50 µM NS1643 for 16 h. ( E ) Quantification of the experiments in ( D ). Error bars indicate the standard error of the mean ( n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Migration, Western Blot, Expressing, Wound Healing Assay

    NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits TGFβ signaling via activation of calcineurin. ( A ) Western blots showing the expression level of phosphorylated smad2/3 and total smad2/3 in HT29 cells treated with DMSO, NS1643 (50 µM) alone, EDTA (10 mM) alone, or NS1643 + EDTA for 2 h. Bar graphs indicate quantification of the experiments in ( A ). Data = mean ± SEM; n = 3; ** p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Activation Assay, Western Blot, Expressing

    NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 abolishes the TGFβ1 effects on R-SMADs phosphorylation and EMT markers ( A ) Western blot showing expression of phosphorylated smad2/3 (P-Smad2/3) in HT29 cells treated for different times as indicated with NS1643 (50 µM) alone, TGFβ1 (2 ng/mL), or NS1643 and TGFβ1. Cells were in a serum-deprived medium for 16 h before application of drugs. Bar graphs indicate quantification of the experiments in ( A ) at the time point of 2 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Western Blot, Expressing

    PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: PP2B-γ Mediates the Inhibitory Effect of NS1643 on TGFβ Signaling. ( A ) Kaplan–Meier plots of overall survival in colon cancer patients with high (pink) and low (blue) expression of PPP3CA, ( B ) PPP3CB, or ( C ) PPP3CC. Side panel indicate the fragments per kilobase of transcript per million (FPKM) mapped reads. ( D ) Survival analysis of PPP3CA, PPP3CB, or PPP3CC gene expression in colorectal cancer patients. ( E ) Western blot showing the expression level of PP2B and E-cadherin at different time after that cells were transfected with si-RNA targeting PPP3CC. For the original Western blots, see Supplementary Figure S1 .

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Western Blot, Transfection

    NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits expression of EMT markers. ( A ) Relative expression level of mRNA encoding for the EMT markers (SNAIL, SLUG, TWIST, and ZEB1) by RT-PCR in HT29, SW480, and FET cells treated with or without NS1643 (50 µM) for 48 h. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p

    Journal: Cancers

    Article Title: Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFβ Signaling via Activation of Calcineurin

    doi: 10.3390/cancers13236025

    Figure Lengend Snippet: NS1643 inhibits R-SMADs phosphorylation ( A ) Western blot showing expression of phosphorylated SMAD2 (P-Smad2) in HT29, ( B ) SW480, or ( C ) FET colon cancer cell lines treated for different times, as indicated with NS1643 (50 µM). Cells were kept in full medium. Bar graph indicates quantification. Data = mean ± SEM; n = 3; * p

    Article Snippet: E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Western Blot, Expressing

    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p

    Journal: PLoS ONE

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    doi: 10.1371/journal.pone.0006168

    Figure Lengend Snippet: Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p

    Article Snippet: Different antagonists (all from Sigma-Aldrich GmbH if not stated otherwise) were dissolved in this bathing solution: 4-aminopyridine (4-AP, 0.1–10 mM), phrixotoxin-1 (PTX, 1–1000 nM, Alomone Labs, Jerusalem, Israel), ammonium chloride (NH4 Cl, 1–100 mM), quinidine (QND, 0.1–100 µM), α-dendrotoxin (DTX, 1–1000 nM), margatoxin (MTX, 0.1–50 nM) and tetraethylammonium chloride (TEA, 1–100 mM).

    Techniques: Inhibition, BrdU Incorporation Assay

    Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p

    Journal: PLoS ONE

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    doi: 10.1371/journal.pone.0006168

    Figure Lengend Snippet: Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p

    Article Snippet: Different antagonists (all from Sigma-Aldrich GmbH if not stated otherwise) were dissolved in this bathing solution: 4-aminopyridine (4-AP, 0.1–10 mM), phrixotoxin-1 (PTX, 1–1000 nM, Alomone Labs, Jerusalem, Israel), ammonium chloride (NH4 Cl, 1–100 mM), quinidine (QND, 0.1–100 µM), α-dendrotoxin (DTX, 1–1000 nM), margatoxin (MTX, 0.1–50 nM) and tetraethylammonium chloride (TEA, 1–100 mM).

    Techniques: Inhibition, MTT Assay

    Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .

    Journal: PLoS ONE

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    doi: 10.1371/journal.pone.0006168

    Figure Lengend Snippet: Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .

    Article Snippet: Different antagonists (all from Sigma-Aldrich GmbH if not stated otherwise) were dissolved in this bathing solution: 4-aminopyridine (4-AP, 0.1–10 mM), phrixotoxin-1 (PTX, 1–1000 nM, Alomone Labs, Jerusalem, Israel), ammonium chloride (NH4 Cl, 1–100 mM), quinidine (QND, 0.1–100 µM), α-dendrotoxin (DTX, 1–1000 nM), margatoxin (MTX, 0.1–50 nM) and tetraethylammonium chloride (TEA, 1–100 mM).

    Techniques: Inhibition, Mass Spectrometry

    Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.

    Journal: PLoS ONE

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    doi: 10.1371/journal.pone.0006168

    Figure Lengend Snippet: Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.

    Article Snippet: Different antagonists (all from Sigma-Aldrich GmbH if not stated otherwise) were dissolved in this bathing solution: 4-aminopyridine (4-AP, 0.1–10 mM), phrixotoxin-1 (PTX, 1–1000 nM, Alomone Labs, Jerusalem, Israel), ammonium chloride (NH4 Cl, 1–100 mM), quinidine (QND, 0.1–100 µM), α-dendrotoxin (DTX, 1–1000 nM), margatoxin (MTX, 0.1–50 nM) and tetraethylammonium chloride (TEA, 1–100 mM).

    Techniques: Patch Clamp, Activation Assay, Mass Spectrometry