ns8593  (Alomone Labs)


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    Alomone Labs ns8593
    Ns8593, 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
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    ns8593  (Alomone Labs)


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    Alomone Labs ns8593
    Ns8593, 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
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    inhibitors  (Alomone Labs)


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    Alomone Labs inhibitors
    Inhibitors, 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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    trpm7 inhibitor ns 8593  (Alomone Labs)


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    Alomone Labs trpm7 inhibitor ns 8593
    Trpm7 Inhibitor Ns 8593, 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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    ns8593  (Alomone Labs)


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    Alomone Labs ns8593
    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 <t>NS8593</t> (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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
    Ns8593, 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/ns8593/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    ns8593 - by Bioz Stars, 2023-01
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    1) Product Images from "TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.606893

    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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
    Figure Legend 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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.

    Techniques Used: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Chemotaxis Assay, Transwell Assay

    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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.
    Figure Legend 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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.

    Techniques Used: Activity Assay, Incubation, Flow Cytometry, Plex 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.
    Figure Legend 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.

    Techniques Used: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Transwell Assay, Injection, Flow Cytometry

    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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).
    Figure Legend 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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).

    Techniques Used: Activity Assay, Plex Assay, Derivative Assay, Incubation, Chemotaxis Assay, Activation Assay

    ns8593  (Alomone Labs)


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    Alomone Labs ns8593
    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 <t>NS8593</t> (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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
    Ns8593, 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/ns8593/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ns8593 - by Bioz Stars, 2023-01
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    Images

    1) Product Images from "TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.606893

    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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
    Figure Legend 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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.

    Techniques Used: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Chemotaxis Assay, Transwell Assay

    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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.
    Figure Legend 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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.

    Techniques Used: Activity Assay, Incubation, Flow Cytometry, Plex 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.
    Figure Legend 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.

    Techniques Used: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Transwell Assay, Injection, Flow Cytometry

    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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).
    Figure Legend 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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).

    Techniques Used: Activity Assay, Plex Assay, Derivative Assay, Incubation, Chemotaxis Assay, Activation Assay

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    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 <t>NS8593</t> (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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
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    1) Product Images from "TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.606893

    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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.
    Figure Legend 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 < 0.05, one-way ANOVA. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of neutrophil chemotaxis toward a CXCL8 gradient (10 nM) in a transwell assay. Human neutrophils were pretreated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) or dissolvent (Ctrl, black), for 30 min prior to CXCL8 or saline (Ctrl) exposure. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, **p < 0.01.

    Techniques Used: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Chemotaxis Assay, Transwell Assay

    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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.
    Figure Legend 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 < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, 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 prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.

    Techniques Used: Activity Assay, Incubation, Flow Cytometry, Plex 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.
    Figure Legend 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 < 0.05, **p < 0.01. Note: there is no difference in TRPM7 current development or amplitude between the two genotypes. (C) Representative dot plot analysis (left and middle panel) and quantification (right panel) of murine neutrophil chemotaxis toward a CXCL1 gradient (10 nM) in a transwell assay. Two-way repeated measurements ANOVA, Sidak’s multiple comparison, *p < 0.05, ***p < 0.001. (D) Murine TNF-α peritonitis model. Saline (Ctrl), or TNF-α (500 ng) were injected intra-peritoneally into Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Numbers of recruited neutrophils in the peritoneum were assessed 4 h later (n = 4–6 mice per group) using flow cytometry. Representative dot plot analyses of recruited neutrophils (left panel) and quantification (right panel). Data are shown as mean ± s.e.m. Statistics: two-way ANOVA, Sidak’s multiple comparison. ***p ≤ 0.001.

    Techniques Used: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay, Transwell Assay, Injection, Flow Cytometry

    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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).
    Figure Legend 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 < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFκB activation was shown to depend on TRPM7 . We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFκB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFκB and mTOR signaling pathways (blue dashed arrow).

    Techniques Used: Activity Assay, Plex Assay, Derivative Assay, Incubation, Chemotaxis Assay, Activation Assay

    ns 8593  (Alomone Labs)


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    Alomone Labs ns 8593
    Effect of TRPM7 inhibitor <t>NS-8593</t> on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution
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    1) Product Images from "Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes"

    Article Title: Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

    Journal: Reproductive Biology and Endocrinology : RB&E

    doi: 10.1186/s12958-020-00643-7

    Effect of TRPM7 inhibitor NS-8593 on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution
    Figure Legend Snippet: Effect of TRPM7 inhibitor NS-8593 on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution

    Techniques Used: Activation Assay, Fluorescence, Inhibition

    Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable
    Figure Legend Snippet: Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable

    Techniques Used:

    ns 8593  (Alomone Labs)


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    Alomone Labs ns 8593
    Ns 8593, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ns8593  (Alomone Labs)


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    Chemical genetics screen identifies pathways that inhibit intracellular growth of B. neotomae. (A) Inhibitory effects of selected small molecules on intracellular replication in J774A.1 host cells at 48 h postinfection. Both B. neotomae (Bn) luminescence and CFU were measured in parallel. Data shown are means ± SD from at least two independent experiments, 3 replicates for luminescence data and 2 replicates for CFU data. Concentrations of the following tested compounds were in general above the IC50 (Table 1) and substantially below the CC50: arcyriaflavin A (3.6 μM), BX912 (3.6 μM), IKK16 (3.2 μM), I3M (3.6 μM), Ki20227 (2.0 μM), CYT387 (1.4 μM), Rhodblock6 (6.9 μM), SP600125 (10.2 μM), esomeprazole (10.2 μM), <t>NS8593</t> (4.7 μM), and pinacidil (10.2 μM). (B) Inhibitory effects of selected small molecules on intracellular replication in primary murine bone marrow-derived macrophages (BMDM) at 48 h postinfection based on luminescence signals. Data shown are means ± SD from 4 replicates. Saponin (0.2%) and 0.3% DMSO in the absence of infection were used as positive and negative cytotoxicity controls, respectively. Significant suppression of luminescence was observed for all inhibitors in comparison with the DMSO control (P < 0.0001 by ANOVA and Dunnett’s post hoc multiple-comparison tests). (C) Effect of kinase target siRNA on intracellular growth of B. neotomae in J774A.1 host cells as assessed by luminescence. Data points represent means ± SD from at least three independent experiments. Significant suppression of luminescence (*, P < 0.001) was observed for siRNA knockdowns compared to the NTsi control. (D) Effect of siRNA knockdowns on mRNA expression assayed using same experimental protocol as the one described for panel C. Data points represent means ± SD from three independent samples and were normalized to the β-actin level within each sample and then to expression in untreated controls.
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    1) Product Images from "A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages"

    Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00044-19

    Chemical genetics screen identifies pathways that inhibit intracellular growth of B. neotomae. (A) Inhibitory effects of selected small molecules on intracellular replication in J774A.1 host cells at 48 h postinfection. Both B. neotomae (Bn) luminescence and CFU were measured in parallel. Data shown are means ± SD from at least two independent experiments, 3 replicates for luminescence data and 2 replicates for CFU data. Concentrations of the following tested compounds were in general above the IC50 (Table 1) and substantially below the CC50: arcyriaflavin A (3.6 μM), BX912 (3.6 μM), IKK16 (3.2 μM), I3M (3.6 μM), Ki20227 (2.0 μM), CYT387 (1.4 μM), Rhodblock6 (6.9 μM), SP600125 (10.2 μM), esomeprazole (10.2 μM), NS8593 (4.7 μM), and pinacidil (10.2 μM). (B) Inhibitory effects of selected small molecules on intracellular replication in primary murine bone marrow-derived macrophages (BMDM) at 48 h postinfection based on luminescence signals. Data shown are means ± SD from 4 replicates. Saponin (0.2%) and 0.3% DMSO in the absence of infection were used as positive and negative cytotoxicity controls, respectively. Significant suppression of luminescence was observed for all inhibitors in comparison with the DMSO control (P < 0.0001 by ANOVA and Dunnett’s post hoc multiple-comparison tests). (C) Effect of kinase target siRNA on intracellular growth of B. neotomae in J774A.1 host cells as assessed by luminescence. Data points represent means ± SD from at least three independent experiments. Significant suppression of luminescence (*, P < 0.001) was observed for siRNA knockdowns compared to the NTsi control. (D) Effect of siRNA knockdowns on mRNA expression assayed using same experimental protocol as the one described for panel C. Data points represent means ± SD from three independent samples and were normalized to the β-actin level within each sample and then to expression in untreated controls.
    Figure Legend Snippet: Chemical genetics screen identifies pathways that inhibit intracellular growth of B. neotomae. (A) Inhibitory effects of selected small molecules on intracellular replication in J774A.1 host cells at 48 h postinfection. Both B. neotomae (Bn) luminescence and CFU were measured in parallel. Data shown are means ± SD from at least two independent experiments, 3 replicates for luminescence data and 2 replicates for CFU data. Concentrations of the following tested compounds were in general above the IC50 (Table 1) and substantially below the CC50: arcyriaflavin A (3.6 μM), BX912 (3.6 μM), IKK16 (3.2 μM), I3M (3.6 μM), Ki20227 (2.0 μM), CYT387 (1.4 μM), Rhodblock6 (6.9 μM), SP600125 (10.2 μM), esomeprazole (10.2 μM), NS8593 (4.7 μM), and pinacidil (10.2 μM). (B) Inhibitory effects of selected small molecules on intracellular replication in primary murine bone marrow-derived macrophages (BMDM) at 48 h postinfection based on luminescence signals. Data shown are means ± SD from 4 replicates. Saponin (0.2%) and 0.3% DMSO in the absence of infection were used as positive and negative cytotoxicity controls, respectively. Significant suppression of luminescence was observed for all inhibitors in comparison with the DMSO control (P < 0.0001 by ANOVA and Dunnett’s post hoc multiple-comparison tests). (C) Effect of kinase target siRNA on intracellular growth of B. neotomae in J774A.1 host cells as assessed by luminescence. Data points represent means ± SD from at least three independent experiments. Significant suppression of luminescence (*, P < 0.001) was observed for siRNA knockdowns compared to the NTsi control. (D) Effect of siRNA knockdowns on mRNA expression assayed using same experimental protocol as the one described for panel C. Data points represent means ± SD from three independent samples and were normalized to the β-actin level within each sample and then to expression in untreated controls.

    Techniques Used: Derivative Assay, Infection, Expressing

    Compound potency, cytotoxicity, and selectivity
    Figure Legend Snippet: Compound potency, cytotoxicity, and selectivity

    Techniques Used:

    Strains and key resources used in this study a
    Figure Legend Snippet: Strains and key resources used in this study a

    Techniques Used: Bicinchoninic Acid Protein Assay, Transfection, Software

    ns 8593  (Alomone Labs)


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    Alomone Labs ns 8593
    Endothelial cell barrier permeability following treatment with a) different concentrations of EtOH or b) the TRPM7 antagonists, NS8593, CyPPA, or SKA-31. *p < 0.05; **p < 0.01. a Mouse BMVECs at Passage 7 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier. The same number of wells without cells were used as background. The wells were then treated with 0 mM, 17.4 mM, 52.2 mM and 104.4 mM EtOH for 24 h, and to determine permeability, 50 ng/μl Evans Blue Dye (EBD) was added into the inserts at the same time and the model was incubated at 37 °C for 24 h. The permeability coefficient was then calculated. P-value was calculated using student’s t test. b Mouse BMVECs at Passage 5 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier (BBB). Wells were then treated with three different Trpm 7 Channel Blockers, NS 8593, CyPPA and SKA-31 at 50 μM for 24 h, and the permeability assay was then performed to determine the effect of TRPM7 blockers on the BBB. P-values were calculated using student’s t test
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    1) Product Images from "Ethanol’s Effects on Transient Receptor Potential Channel Expression in Brain Microvascular Endothelial Cells"

    Article Title: Ethanol’s Effects on Transient Receptor Potential Channel Expression in Brain Microvascular Endothelial Cells

    Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

    doi: 10.1007/s11481-018-9796-3

    Endothelial cell barrier permeability following treatment with a) different concentrations of EtOH or b) the TRPM7 antagonists, NS8593, CyPPA, or SKA-31. *p < 0.05; **p < 0.01. a Mouse BMVECs at Passage 7 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier. The same number of wells without cells were used as background. The wells were then treated with 0 mM, 17.4 mM, 52.2 mM and 104.4 mM EtOH for 24 h, and to determine permeability, 50 ng/μl Evans Blue Dye (EBD) was added into the inserts at the same time and the model was incubated at 37 °C for 24 h. The permeability coefficient was then calculated. P-value was calculated using student’s t test. b Mouse BMVECs at Passage 5 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier (BBB). Wells were then treated with three different Trpm 7 Channel Blockers, NS 8593, CyPPA and SKA-31 at 50 μM for 24 h, and the permeability assay was then performed to determine the effect of TRPM7 blockers on the BBB. P-values were calculated using student’s t test
    Figure Legend Snippet: Endothelial cell barrier permeability following treatment with a) different concentrations of EtOH or b) the TRPM7 antagonists, NS8593, CyPPA, or SKA-31. *p < 0.05; **p < 0.01. a Mouse BMVECs at Passage 7 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier. The same number of wells without cells were used as background. The wells were then treated with 0 mM, 17.4 mM, 52.2 mM and 104.4 mM EtOH for 24 h, and to determine permeability, 50 ng/μl Evans Blue Dye (EBD) was added into the inserts at the same time and the model was incubated at 37 °C for 24 h. The permeability coefficient was then calculated. P-value was calculated using student’s t test. b Mouse BMVECs at Passage 5 were seeded into Corning transwell inserts seated in 24-well plates to construct an in-vitro Blood-Brain Barrier (BBB) model. The cells were allowed to grow in the inserts for three days to form a barrier (BBB). Wells were then treated with three different Trpm 7 Channel Blockers, NS 8593, CyPPA and SKA-31 at 50 μM for 24 h, and the permeability assay was then performed to determine the effect of TRPM7 blockers on the BBB. P-values were calculated using student’s t test

    Techniques Used: Permeability, Construct, In Vitro, Incubation

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    Effect of TRPM7 inhibitor <t>NS-8593</t> on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution
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    Effect of TRPM7 inhibitor NS-8593 on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution

    Journal: Reproductive Biology and Endocrinology : RB&E

    Article Title: Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

    doi: 10.1186/s12958-020-00643-7

    Figure Lengend Snippet: Effect of TRPM7 inhibitor NS-8593 on oocyte activation. a [Ca2+]i oscillations of NS-8593-inhibited oocytes. b Mitochondrial membrane potential dynamic changes of 1 μM NS-8593-inhibited oocytes. c Mitochondrial membrane potential fluorescence intensity of 1 μM NS-8593-inhibited oocytes. The green and red curves represent low and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM NS-8593 inhibition of oocyte spindle formation e Living cell observation of 1 μM NS-8593 inhibition of oocyte subcortical F-actin distribution

    Article Snippet: The details of Ca 2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca 2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

    Techniques: Activation Assay, Fluorescence, Inhibition

    Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable

    Journal: Reproductive Biology and Endocrinology : RB&E

    Article Title: Regulation of [Ca 2+ ] i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

    doi: 10.1186/s12958-020-00643-7

    Figure Lengend Snippet: Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p < 0.05 (*) was considered statistically significant. (X) indicates data unavailable

    Article Snippet: The details of Ca 2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca 2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

    Techniques: