ns8593  (Alomone Labs)

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

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

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

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