trpm7 inhibitor  (MedChemExpress)

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) TRPM7 current densities and B) TRPM7 I/V relationship of Jurkat cells during whole-cell patch clamp experiment with Mg 2+ -free intracellular solution. WT (WT, black) and TRPM7 KO Jurkat clones (KO, red), n(WT)=9; n(KO)=10. C) Cell counts and D) viability of natively proliferating TRPM7 WT and KO Jurkat clones in RPMI medium with 10% FBS, with and without supplementation with 6 mM MgCl 2 , n=3, measured in duplicates. E) Cellular Mg 2+ content quantified by ICP-MS. WT and TRPM7 KO Jurkat clones, cultured in regular (WT-)media for 18 h ahead of sampling, n=4. And WT and TRPM7 KO Jurkat clones, cultured in regular (WT-)media supplemented with 6 mM MgCl 2 for 18 h ahead of sampling, n=4. F) TRPM7 current densities and G) TRPM7 I/V relationship of Jurkat cells during whole-cell patch clamp with Mg 2+ -free intracellular solution. WT Jurkat cells, treated with DMSO as solvent control (Ctrl, black) or treated with 30 µM NS8593 (NS, red), n(Ctrl)=6; n(NS)=10. H) Cell counts and I) viability of natively proliferating Jurkat cells in RPMI medium with 10% FBS, with and without supplementation with 6 mM MgCl 2 , and treated with DMSO as solvent control (Ctrl, black) or treated with 30 µM NS8593 (NS, red), n=4. J) Cellular Mg 2+ content as measured with ICP-MS. Jurkat WT cells, treated with DMSO as solvent control (Ctrl, black) or treated with 30 µM NS8593 in DMSO (NS, red), cultured in regular (WT-) media without and with supplementation with 6 mM MgCl 2 for 18 h ahead of sampling, n=4. Statistics: Two-way ANOVA (C, D, H, I) or one-way ANOVA (E, J). * P<0.05; ** P<0.005; *** P<0.0005 and **** P<0.0001. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Patch Clamp, Clone Assay, Cell Culture, Sampling, Solvent, Control

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) TRPM7 current densities and B) TRPM7 I/V relationship of Jurkat cells during whole-cell patch clamp experiment with Mg 2+ -free intracellular solution. WT (WT, grey) and TRPM7 KO2 Jurkat clone (KO2, orange). n(WT)=9; n(KO2)=10. C) Cell counts and D) viability of natively proliferating TRPM7 WT and KO2 Jurkat clone in RPMI medium with 10% FBS, with and without supplementation with 6 mM MgCl 2 . n=3, measured in duplicates. E) Cellular Mg contents quantified by ICP-MS. WT and TRPM7 KO2 Jurkat clone, cultured in regular (WT-)media or in medium supplemented with 6 mM MgCl 2 for 18 h ahead of sampling, n=4. F) Fura-2 based imaging of cytosolic Ca 2+ concentration of Jurkat cells. Stimulation with 5 µM thapsigargin at indicated time point (arrow). WT (WT, grey) and TRPM7 KO2 (KO2, orange) Jurkat clone, n (WT) =111; n (KO2) = 59; G) Quantification of the area under the curve (AUC) of respective curves shown in F. H) Representative immuno-fluorescent images of NFATc1 localization in WT and KO2 clone before (basal) and after 30 min stimulation (stim.) with 5 µM thapsigargin, scale bar = 2 μm. NFATc1 in red, DAPI in blue. I) Quantification of nuclear NFATc1 levels upon stimulation of TRPM7 WT (WT, grey) and KO (KO2, orange) clone. n (WT) = 261; n (KO2) = 149. Statistics: Two-way ANOVA (C, D), one-way ANOVA (E) or Student’s t test (G, I). * P<0.05; and **** P<0.0001. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Patch Clamp, Cell Culture, Sampling, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) TRPM7 current densities and B) TRPM7 I/V relationship of Jurkat T cells during whole-cell patch clamp experiment with Mg 2+ -free intracellular solution. Controls (Ctrl, grey) and cells treated with 1 µM apamin (Apamin, blue), n (Ctrl)=9, n (Apamin)=6. C) Cell counts and D) viability of natively proliferating Jurkat cells in RPMI medium with 10% FBS, with and without 1 µM apamin (Apamin, blue), n=4. E) Flow cytometry of upregulation of activation markers CD69 in primary CD4 T-lymphocytes 48 h after anti-CD3/CD28 stimulation. Cells treated either as control (Ctrl, grey) or with 1 µM apamin (Apamin, blue), n=4. F) Representative trace of CD4 T cells Fura-2 based imaging of cytosolic Ca 2+ concentrations following anti-CD3/CD28 stimulation. Antibodies bound to microscopy chamber bottom with cells sinking down in saline containing 2 mM Ca 2+ during running measurement, coming to rest in focus plane with contact to stimulation antibodies. Cells measured as control (Ctrl, grey) or in presence of 1 µM apamin (Apamin, blue). Statistics: Student’s t test (D). n.s.—not significant. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Patch Clamp, Flow Cytometry, Activation Assay, Control, Imaging, Microscopy, Saline

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) Fura-2 based imaging of cytosolic Ca 2+ concentration of Jurkat cells. Stimulation with 5 µM thapsigargin at the indicated time point (arrow) of WT (WT, black) and TRPM7 KO (KO, red) Jurkat cells, n(WT)=111; n(KO)=113. B) Quantification of the area under the curve (AUC) of respective curves shown in A. C) Representative immune-fluorescent images of the NFATc1 localization in WT and KO cells before (basal) and after 30 min stimulation (stim.) with 5 µM thapsigargin, scale bar = 2 μm. NFATc1 in red, DAPI in blue. D) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (WT, black) and KO (KO, red) cells, n(WT)= 261; n(KO)=279. E) Relative IL-2 mRNA expression levels of Jurkat WT (WT, black) and KO (Ko, red) cells, n=4. F) CD69 expression of stimulated Jurkat cells, WT (WT, black) and KO (KO, red), n=5. G) Ca 2+ imaging of WT Jurkat, treated with DMSO as solvent control cells (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red). Stimulation with 5 µM thapsigargin at indicated time point (arrow), n(Ctrl)=95; n(NS)=94. H) Quantification of the area under the curve (AUC) of respective curves shown in G. I) Representative immune-fluorescent images of NFATc1 localization in DMSO treated cells as solvent control (Ctrl, black) or treated cells with 30 µM NS8593 (NS, red) before and after 30 min stimulation with 5 µM thapsigargin, scale bar = 2 μm. J) Quantification of nuclear NFATc1 levels upon stimulation of cells treated with DMSO as solvent control (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red), n(Ctrl)=196; n(NS)=195. K) Relative IL-2 mRNA expression levels of cells treated with DMSO as solvent control (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red), n=7. M) CD69 expression of cells treated with DMSO as solvent control (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red), after α-CD3 stimulated, n=6-7. Statistics: Student’s t test (B, D, F, H, I, M) and Mann-Whitney U test (E, K). ** P<0.005; *** P<0.0005; **** P<0.0001 and n.s.—not significant. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Imaging, Concentration Assay, Expressing, Solvent, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) TRPM7 I/V relationship of naïve CD4 T cells during whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells treated with DMSO as solvent control (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red). B) Representative trace of naïve CD4 T cells Fura-2 based imaging of cytosolic Ca 2+ concentrations following anti-CD3/CD28 stimulation. Antibodies bound to microscopy chamber bottom with cells sinking down in saline containing 2 mM Ca 2+ during running measurement, coming to rest in focus plane with contact to stimulation antibodies. Cells treated with DMSO as solvent control (Ctrl, black) or treated with 30 µM NS8593 (NS, red). Respective quantification of Ca 2+ imaging experiments of naïve CD4 T cells for C) basal, D) delta Ca 2+ , E) AUC and F) oscillation frequency, n=29-30 cells. G) TRPM7 I/V relationship of conventional CD4 T cells during whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells treated with DMSO as solvent control (Ctrl, black) or cells treated with 30 µM NS8593 (NS, red), n(Ctrl)=5, n(NS)=5. H) Representative trace of conventional CD4 T cells Fura-2 based imaging of cytosolic Ca 2+ concentrations following anti-CD3/CD28 stimulation. Antibodies bound to microscopy chamber bottom with cells sinking down in saline containing 2 mM Ca 2+ during running measurement, coming to rest in focus plane with contact to stimulation antibodies. Cells treated with DMSO as solvent control (Ctrl, black) or treated with 30 µM NS8593 (NS, red). Respective quantification of Ca 2+ imaging experiments of conventional CD4 T cells for I) basal, J) delta Ca 2+ , K) AUC and L) oscillation frequency, n= 39-48 cells. M) Representative immune-fluorescent images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profile of subcellular NFATc1 distribution (Ctrl in black, NS in red, respective DAPI in blue) of naïve CD4 T cells treated with DMSO as solvent control and TRPM7 inhibited cells upon 30 min stimulation with anti-CD3/CD28, scale bar = 2 μm. N) Quantification of nuclear NFATc1 levels upon stimulation of cells treated with DMSO as solvent (Ctrl, black) or in presence of 30 µM NS8593 (NS, red) cells, n(Ctrl)=149; n(NS)=144. O) Representative immune-fluorescent images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profile of subcellular NFATc1 distribution (Ctrl in black, NS in red, respective DAPI in blue) of conventional CD4 T cells of Ctrl and TRPM7-inhibited cells upon 30 min stimulation with anti-CD3/CD28, scale bar = 2 μm. NFATc1 in red, DAPI in blue. P) Quantification of nuclear NFATc1 levels upon stimulation and treatment with DMSO as solvent control (Ctrl, black) or in presence of 30 µM NS8593 (NS, red) cells, n(Ctrl)=155; n(NS)=132. Statistics: Student’s t test (C-F, I-L, N, P). * P<0.05; **** P<0.0001 and n.s.—not significant. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Patch Clamp, Solvent, Control, Imaging, Microscopy, Saline

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) IL-2 quantification of supernatant of naïve CD4 T cells 48 h after anti-CD3/CD28 stimulation, n=4-5. Histograms and quantification of upregulated activation markers CD69 (B-C) and CD25 (D-E) in naïve CD4 T lymphocytes 48 h after stimulation. Cells treated either with DMSO as solvent control (Ctrl, black) or with 30 μM NS8593 (NS, red), both with and without supplementation with 6 mM MgCl 2 . F) IL-2 quantification of supernatant of conventional CD4 T cells 48 h after anti-CD3/CD28 stimulation or cells treated with DMSO as solvent control (Ctrl, black) or with 30 µM NS8593 (NS, red), both with and without supplementation with 6 mM MgCl 2 , n=4-5. Histograms and quantification of upregulated activation markers CD69 (G-H) and CD25 (I-J) in conventional CD4 T lymphocytes 48 h after stimulation. Cells treated either with DMSO as solvent control (Ctrl, black) or 30 μM NS8593 (NS, red), both with and without supplementation with 6 mM MgCl 2 . K) TRPM7 I/V relationship of conventional CD4 T cells during whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells treated with EtOH as solvent control (Ctrl, black) or cells treated with 10 µM waixenicinA (WxA, green). Histograms and quantification of upregulated activation markers CD69 (L-M) and CD25 (N-O) in conventional CD4 T lymphocytes 48 h after stimulation. Cells treated either with EtOH as solvent control (Ctrl, black) or 10 μM waixenicinA (WxA, green), both with and without supplementation of 6 mM MgCl 2 , n=7. Statistics: One-way ANOVA (A, C, E, F, H, J, M, O). * P<0.05; ** P<0.005; *** P<0.0005; **** P<0.0001 and n.s.—not significant. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Activation Assay, Solvent, Control, Patch Clamp

Journal: bioRxiv

Article Title: TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner

doi: 10.1101/2024.12.04.626765

Figure Lengend Snippet: A) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of conventional CD4 T cells in presence of various NS8593 concentrations, with (right) and without (left) supplementation with 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 cells. Color code as in B. Cells gated on T cell population, single cells and CD4 T cells. B) Respective quantification of NS8593 dose dependent proliferation of conventional CD4 T cells, with and without supplementation with 6 mM MgCl 2 , corresponding to H, n=4-7. C) Representative FACS plots and gating path of iTreg cells after 6 days of differentiation of naïve CD4 T cells, cells treated with DMSO as solvent control (upper panel) or treated with 30 µM NS8593 (lower panel). D) Representative histogram overlay of FOXP3 signal in Boolean gate of DMSO controls (Ctrl, black) or in presence of 30 µM NS8593 (NS, red). E) Respective quantification of FOXP3 signal of cells treated with DMSO as solvent control (Ctrl, black) or 30 µM NS8593 (NS, red), n(Ctrl)=14; n(NS)=8. F) Respective quantification of FOXP3 signal of cells treated with EtOH as solvent control (Ctrl, black), 10 µM waixenicin A (WxA, blue) or 10 µM waixenicin A (WxA, green), n(Ctrl)=14; n(10 µM WxA)=11; n(30 µM WxA)=4. G) Respective quantification of FOXP3 signal of EtOH controls (Ctrl, black), DMSO Ctrl + 6 mM MgCl 2 (Ctrl+MgCl 2 , blue), 30 µM waixenicin A + MgCl 2 (WxA+MgCl 2 , turquoise), n(Ctrl)=20; n(Ctrl+MgCl 2 )=20; n(30 µM WxA + MgCl 2 )=12. H) Graphical summary of TRPM7-independent iT reg differentiation. Pharmacological blockade of TRPM7 reduces intracellular Mg 2+ levels and results in reduced IL-2 secretion, impaired upregulation of T-cell activation markers CD69 and CD25 and diminished proliferation in presence of TCR stimulus. TRPM7 inhibition followed by polarization of naïve CD4 T cells in presence of anti-CD3/CD28, IL-2, TGF-ß and ATRA, an iT reg polarization cocktail, results in lower iT reg numbers but enhanced FOXP3 expression. Figure created in https://BioRender.com . Statistics: One-way ANOVA (B, F, G) and Student’s t test (E). * P<0.05; ** P<0.005; *** P<0.0005; **** P<0.0001 and n.s.—not significant. Data are mean ± SD.

Article Snippet: Synthetic TRPM7 inhibitor NS8395 was purchased from Alomone.

Techniques: Solvent, Control, Activation Assay, Inhibition, Expressing