anti trpm7 alomone labs  (Alomone Labs)

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Sanger sequencing traces of TRPM7 WT and KO gDNA. (B) Relative TRPM7 mRNA expression levels of Jurkat TRPM7 WT (black) and KO clones (KO1, red; KO2, orange), with respect to HPRT levels. Primers were designed to cover the sequence of the one-base pair insertion in exon 4, n = 9. (C) Representative Western blot of TRPM7 expression (black arrow) in Jurkat TRPM7 WT and KO clones. (B) Statistics: One-way ANOVA (B). *** P < 0.0005. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Sequencing, Expressing, Clone Assay, Western Blot

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) TRPM7 current densities extracted at −80 and +80 mV, and (B) representative I/V relationships characteristic for TRPM7 channels in TRPM7 WT (black) and TRPM7 KO (red) Jurkat T-cell clones obtained via whole-cell patch clamp recordings in the absence of intracellular Mg 2+ , n (WT) = 9; n (KO) = 10. (C) Cell counts and (D) viability of proliferating TRPM7 WT (black) and KO (red) Jurkat clones in medium, with or without supplementation of 6 mM MgCl 2 , n = 3, measured in duplicates. (E) Cellular Mg 2+ content quantified by ICP-MS. TRPM7 WT (black) and KO (red) Jurkat T-cell clones cultured in regular media, with or without MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (F, G) TRPM7 current densities extracted at −80 and +80 mV, and (G) representative TRPM7 I/V relationships of TRPM7 WT Jurkat T cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), obtained via whole-cell patch clamp recordings as in (A, B), n (Ctrl) = 6; n (NS) = 10. (H) Cell counts and (I) viability of proliferating Jurkat T cells in medium, with or without supplementation of 6 mM MgCl 2 , treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 4. (J) Cellular Mg 2+ content quantified by ICP-MS. Jurkat WT cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) cultured in regular media with or without 6 mM MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (C, D, E, H, I, J) Statistics: two-way ANOVA (C, D, H, I) or t test (E, J). * P < 0.05; ** P < 0.005; *** P < 0.0005 and **** P < 0.0001. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) 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. TRPM7 WT (WT, gray) and 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 of 6 mM MgCl 2 , n = 3, measured in duplicates. (E) Cellular Mg 2+ contents quantified by ICP-MS. TRPM7 WT and KO2 Jurkat clone, cultured in regular (WT-)media without or with 6 mM MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (F) Fura-2-based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow). TRPM7 WT (WT, gray) and 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 TRPM7 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, J) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) in basal state (I) and upon 30 min passive store depletion induced with 5 μM thapsigargin (J). (K) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (WT, gray) and KO (KO2, orange) clone, n (WT) = 261; n (KO2) = 149. (L) Histograms and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) cells after overnight α-CD3 stimulation, n = 4–6. (B, F, G, H, J) Statistics: One-way ANOVA (B), Two-way ANOVA (F, G), or t test (H, J). * P < 0.05; **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) 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. Control (Ctrl, gray) 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 T cells in RPMI medium with 10% FBS, treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray), n = 4. (E) Cell counts and (F) viability of natively proliferating Jurkat TRPM7 WT and KO cells in RPMI medium with 10% FBS, treated with 30 μM NS8593, 30 μM NS8593 with additional 6 mM MgCl 2 or untreated controls, n = 3, measured in duplicates. (G) Fura-2 based imaging of cytosolic Ca 2+ concentrations of Jurkat TRPM7 WT and KO cells treated with 30 μM NS8593 or left untreated. Passive store depletion was induced with 5 μM thapsigargin at indicated time point (arrow). (H) Quantification of area under the curve (AUC) of traces shown in (G), n = 24–70. (I) Representative FACS plots and gating strategy for CD69 visualization, shown for Jurkat WT cells. (J) Histogram and (K) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT cells treated with 1 μM Apamin (Apamin, blue) compared with untreated controls (Ctrl, light gray), n = 3. (L) Histogram and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT or KO cells treated with 30 μM NS8593 or left untreated, n = 4–6. (H, K, M) Statistics: One-way ANOVA (H, M) and t test (K). ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Patch Clamp, Control, Imaging, Expressing

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Fura-2 based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at the indicated time point (arrow) of WT (black) and TRPM7 KO (red) Jurkat T 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-fluorescence images of the NFATc1 localization in TRPM7 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, E) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (D) and upon 30 min passive store depletion induced with 5 μM thapsigargin (E). (F) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (black) and KO (red) cells, n (WT) = 261; n (KO) = 279. (G) Relative IL-2 mRNA expression levels of Jurkat TRPM7 WT (black) and KO (red) cells, n = 4. (H) Histograms and (I) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (black) and KO (red) cells after overnight stimulation with α-CD3, n = 4–6. (J) Quantification of Ca 2+ signals of TRPM7 WT Jurkat T cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow), n (Ctrl) = 95; n (NS) = 94. (K) Quantification of the area under the curve (AUC) of respective Ca 2+ signals shown in (G). (L) Representative immune-fluorescence images of NFATc1 localization of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) before and after 30 min stimulation with 5 μM thapsigargin, scale bar = 2 μm. (M, N) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (M) and upon 30 min passive store depletion induced with 5 μM thapsigargin (N). (O) Quantification of nuclear NFATc1 levels upon stimulation of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 196; n (NS) = 195. (P) Relative IL- 2 mRNA expression levels of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 7. (Q) Histograms and (R) quantification of up-regulated CD69 expression of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) after α-CD3 stimulation, n = 6–7. (B, D, E, F, H, J, K, M) Statistics: t test (B, D, F, H, J, 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: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative I/V relationships of TRPM7 channels in naïve CD4 T cells obtained via whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (B) Representative single-cell traces of cytosolic Ca 2+ concentrations of naïve CD4 T cells following α-CD3/α-CD28 stimulation. Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) in saline (2 mM CaCl 2 ). (C, D, E, F) Quantification of Ca 2+ signals of naïve CD4 T cells for (C) basal, (D) delta Ca 2+ , (E) AUC and (F) oscillation frequency, n = 39–48 cells. (G) Representative I/V relationships of TRPM7 channels in total CD4 T cells obtained via whole-cell patch clamp using Mg 2+ -free intracellular solution. Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (H) Representative single-cell traces of cytosolic Ca 2+ concentrations of total CD4 T cells following α-CD3/α-CD28 stimulation. Data obtained as in (B). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (I, J, K, L) Quantification of Ca 2+ signals of total CD4 T cells for (I) basal, (J) delta Ca 2+ , (K) AUC, and (L) oscillation frequency, n = 29–30 cells. (M) Representative immune-fluorescence images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profiles of subcellular NFATc1 distribution (Ctrl, black; NS, red; DAPI, light blue) of naïve CD4 T cells treated with 30 μM NS8593 (NS) or DMSO (Ctrl) upon 30 min stimulation with α-CD3/α-CD28, scale bar = 2 μm. (N) Quantification of nuclear NFATc1 levels upon 30 min α-CD3/α-CD28 stimulation of cells treated with 30 μM NS8593 (NS, red) or DMSO (Ctrl, black), n (Ctrl) = 149; n (NS) = 144. (O) Representative immune-fluorescence images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profiles of subcellular NFATc1 distribution (Ctrl, black; NS, red; DAPI, light blue) of total CD4 T cells treated with 30 μM NS8593 (NS) or DMSO (Ctrl) upon 30 min stimulation with α-CD3/α-CD28, scale bar = 2 μm. (P) Quantification of nuclear NFATc1 levels upon 30 min α-CD3/α-CD28 stimulation in presence of 30 μM NS8593 (NS, red) cells or DMSO control (Ctrl, black) or, n (Ctrl) = 155; n (NS) = 132. (C, D, E, F, I, J, K, L, N, P) Statistics: t test (C, D, E, F, I, J, K, L, N, P). * P < 0.05; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Patch Clamp, Control, Saline, Fluorescence

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) IL-2 quantification in supernatant of naïve CD4 T cells 48 h after α-CD3/α-CD28 stimulation, n = 4–5. (B, C, D, E) Histograms and quantification of up-regulated activation markers CD69 (B, C) and CD25 (D, E) in naïve CD4 T lymphocytes 48 h after stimulation. Cells were treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (F) IL-2 quantification in supernatant of total CD4 T cells 48 h after α-CD3/α-CD28 stimulation or cells treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 , n = 4–5. (G, H, I, J) Histograms and quantification of up-regulated activation markers CD69 (G, H) and CD25 (I, J) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with either 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (K) Representative TRPM7 I/V relationships of total CD4 T cells obtained via whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells were treated with 10 μM Waixenicin A (WxA, green) or EtOH control (Ctrl, black). (L, M, N, O) Histograms and quantification of up-regulated activation markers CD69 (L, M) and CD25 (N, O) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with 10 μM Waixenicin A or EtOH control, both with (Ctrl, blue; WxA, light green) and without (Ctrl, black; WxA, green) supplementation of 6 mM MgCl 2 , n = 7. (P) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various NS8593 concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (Q). (Q) Respective quantification of NS8593 dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (P), n = 4–7. (R) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various Waixenicin A concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (S). (S) Respective quantification of Waixenicin A dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (S), n = 4–8. (A, C, E, F, H, J, M, O, Q, S) Statistics: one-way ANOVA (A, C, E, F, H, J, M, O, Q, S). * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Activation Assay, Control, Patch Clamp

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative Western blot of pAKT Ser473 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO (Ctrl) in resting state and after 30 min α-CD3/α-CD28 stimulation. (B) Respective quantification of pAKT Ser473 signals from blots shown in (A), n = 6. (C) Representative immune-fluorescence images of AKT1-TRPM7 proximity-ligation interaction in CD4 T cells in resting state or after stimulation with α-CD3/α-CD28 for 30 min. AKT1-TRPM7 interactions are visible as red dots, DAPI in blue, scale bar = 5 μm. (D) Respective quantification of PLA signals per cell upon α-CD3/α-CD28 stimulation as shown in (C). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 173; n (NS) = 115. (E) Representative Western blot of pSMAD2 Ser465/Ser467 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min TGF-β stimulation. (F) Respective quantification of pSMAD2 Ser465/Ser467 signals from blots shown in E, n = 6. (G) Histogram and (H) quantification of pSMAD2 Ser465/Ser467 signals in CD4 T cells after 15 min TGF-β stimulation. Cells treated with 30 μM NS8593 (NS) or DMSO control (Ctrl), unstimulated Ctrl shown in light gray, n = 6. (I) Representative Western blot of pSMAD2 Ser465/Ser467 signals in CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 ) or H 2 O control (Ctrl) in resting state and after 10 min TGF-β stimulation. (J) Respective quantification of pSMAD2 Ser465/Ser467 signals from blots shown in (I), n = 4. (K) Histogram and (L) quantification of pSMAD2 Ser465/Ser467 signals in CD4 T cells after 15 min TGF-β stimulation. Cells treated with 6 mM MgCl 2 (MgCl 2 , blue) or H 2 O control (Ctrl, black), unstimulated Ctrl shown in light gray, n = 6–7. (M) Representative immune-fluorescence images of SMAD2-TRPM7 proximity-ligation interaction in CD4 T cells in resting state or after stimulation with TGF-β for 10 min. SMAD2-TRPM7 interactions are visible as red dots, DAPI in blue, scale bar = 5 μm. (N) Respective quantification of PLA signals per cell upon TGF-β stimulation as shown in (M). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 327; n (NS) = 284. (B, D, F, H, J, L, N) Statistics: one-way ANOVA (B, F, J) and t test (D, F, H, L, N). * P < 0.05; ** P < 0.005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Western Blot, Fluorescence, Ligation, Control

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Schematic description of naïve CD4 T-cell differentiation towards FOXP3-expressing regulatory T cells and RORƔt-expressing T H 17 cells, including respective cytokine polarization milieus. (B, C) Percentages of CD45RA − cells and (C) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6–7. (D, E) Representative FACS histograms and (E) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 6–7. (F, G) Percentages of CD45RA − cells and (G) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 7. (H, I) Representative FACS histograms and (I) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (J) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6. (K, L) Representative FACS histograms and (L) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 4–6. (M) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (N, O) Representative FACS histograms and (O) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 5. (P) Graphical summary of TRPM7-(in)dependent T-cell activation and differentiation towards iT reg and iT H 17 cells. Pharmacological blockade of TRPM7 reduces intracellular Mg 2+ levels, leads to reduced Ca 2+ signaling and results in reduced IL-2 secretion, impaired up-regulation of T-cell activation markers CD69 and CD25, and diminished proliferation upon TCR stimulus (left). TRPM7 inhibition during polarization of naïve CD4 T cells into iT reg cells preserves FOXP3 + signals of CD25 + CD127 lo iT reg cells. Polarization of naïve CD4 T cells into iT H 17 cells results in augmented RORƔt expression in the presence of 6 mM Mg 2+ , which is reduced upon TRPM7 inhibition, highlighting the need for Mg 2+ uptake and related TRPM7-dependent intracellular signaling for iT H 17 cell polarization (right). (B, C, E, F, G, I, J, L, M, O) Statistics: one-way ANOVA (B, C, E, J, L) and t test (F, G, I, 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: The following antibodies were used: α-TRPM7 (ACC-047; Alomone) or α-HSP90 (C45G5; Cell Signaling).

Techniques: Cell Differentiation, Expressing, Control, Activation Assay, Inhibition

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Sanger sequencing traces of TRPM7 WT and KO gDNA. (B) Relative TRPM7 mRNA expression levels of Jurkat TRPM7 WT (black) and KO clones (KO1, red; KO2, orange), with respect to HPRT levels. Primers were designed to cover the sequence of the one-base pair insertion in exon 4, n = 9. (C) Representative Western blot of TRPM7 expression (black arrow) in Jurkat TRPM7 WT and KO clones. (B) Statistics: One-way ANOVA (B). *** P < 0.0005. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Sequencing, Expressing, Clone Assay, Western Blot

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) TRPM7 current densities extracted at −80 and +80 mV, and (B) representative I/V relationships characteristic for TRPM7 channels in TRPM7 WT (black) and TRPM7 KO (red) Jurkat T-cell clones obtained via whole-cell patch clamp recordings in the absence of intracellular Mg 2+ , n (WT) = 9; n (KO) = 10. (C) Cell counts and (D) viability of proliferating TRPM7 WT (black) and KO (red) Jurkat clones in medium, with or without supplementation of 6 mM MgCl 2 , n = 3, measured in duplicates. (E) Cellular Mg 2+ content quantified by ICP-MS. TRPM7 WT (black) and KO (red) Jurkat T-cell clones cultured in regular media, with or without MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (F, G) TRPM7 current densities extracted at −80 and +80 mV, and (G) representative TRPM7 I/V relationships of TRPM7 WT Jurkat T cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), obtained via whole-cell patch clamp recordings as in (A, B), n (Ctrl) = 6; n (NS) = 10. (H) Cell counts and (I) viability of proliferating Jurkat T cells in medium, with or without supplementation of 6 mM MgCl 2 , treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 4. (J) Cellular Mg 2+ content quantified by ICP-MS. Jurkat WT cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) cultured in regular media with or without 6 mM MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (C, D, E, H, I, J) Statistics: two-way ANOVA (C, D, H, I) or t test (E, J). * P < 0.05; ** P < 0.005; *** P < 0.0005 and **** P < 0.0001. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) 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. TRPM7 WT (WT, gray) and 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 of 6 mM MgCl 2 , n = 3, measured in duplicates. (E) Cellular Mg 2+ contents quantified by ICP-MS. TRPM7 WT and KO2 Jurkat clone, cultured in regular (WT-)media without or with 6 mM MgCl 2 supplementation for 18 h ahead of sampling, n = 4. (F) Fura-2-based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow). TRPM7 WT (WT, gray) and 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 TRPM7 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, J) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) in basal state (I) and upon 30 min passive store depletion induced with 5 μM thapsigargin (J). (K) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (WT, gray) and KO (KO2, orange) clone, n (WT) = 261; n (KO2) = 149. (L) Histograms and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (WT, gray) and KO2 (KO2, orange) cells after overnight α-CD3 stimulation, n = 4–6. (B, F, G, H, J) Statistics: One-way ANOVA (B), Two-way ANOVA (F, G), or t test (H, J). * P < 0.05; **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A, B) 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. Control (Ctrl, gray) 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 T cells in RPMI medium with 10% FBS, treated with 1 μM Apamin (Apamin, blue) or control (Ctrl, gray), n = 4. (E) Cell counts and (F) viability of natively proliferating Jurkat TRPM7 WT and KO cells in RPMI medium with 10% FBS, treated with 30 μM NS8593, 30 μM NS8593 with additional 6 mM MgCl 2 or untreated controls, n = 3, measured in duplicates. (G) Fura-2 based imaging of cytosolic Ca 2+ concentrations of Jurkat TRPM7 WT and KO cells treated with 30 μM NS8593 or left untreated. Passive store depletion was induced with 5 μM thapsigargin at indicated time point (arrow). (H) Quantification of area under the curve (AUC) of traces shown in (G), n = 24–70. (I) Representative FACS plots and gating strategy for CD69 visualization, shown for Jurkat WT cells. (J) Histogram and (K) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT cells treated with 1 μM Apamin (Apamin, blue) compared with untreated controls (Ctrl, light gray), n = 3. (L) Histogram and (M) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT or KO cells treated with 30 μM NS8593 or left untreated, n = 4–6. (H, K, M) Statistics: One-way ANOVA (H, M) and t test (K). ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s., not significant. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Patch Clamp, Control, Imaging, Expressing

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Fura-2 based imaging of cytosolic Ca 2+ concentration of Jurkat T cells. Passive store release was induced with 5 μM thapsigargin at the indicated time point (arrow) of WT (black) and TRPM7 KO (red) Jurkat T 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-fluorescence images of the NFATc1 localization in TRPM7 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, E) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (D) and upon 30 min passive store depletion induced with 5 μM thapsigargin (E). (F) Quantification of nuclear NFATc1 levels (corresponding to AF647 signal intensity) upon stimulation of TRPM7 WT (black) and KO (red) cells, n (WT) = 261; n (KO) = 279. (G) Relative IL-2 mRNA expression levels of Jurkat TRPM7 WT (black) and KO (red) cells, n = 4. (H) Histograms and (I) quantification of up-regulated CD69 expression of Jurkat TRPM7 WT (black) and KO (red) cells after overnight stimulation with α-CD3, n = 4–6. (J) Quantification of Ca 2+ signals of TRPM7 WT Jurkat T cells, treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). Passive store release was induced with 5 μM thapsigargin at indicated time point (arrow), n (Ctrl) = 95; n (NS) = 94. (K) Quantification of the area under the curve (AUC) of respective Ca 2+ signals shown in (G). (L) Representative immune-fluorescence images of NFATc1 localization of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) before and after 30 min stimulation with 5 μM thapsigargin, scale bar = 2 μm. (M, N) Representative intensity profiles of subcellular NFATc1 localization (NFATc1 in red, DAPI in blue) of Jurkat TRPM7 WT (black) and KO (red) cells in basal state (M) and upon 30 min passive store depletion induced with 5 μM thapsigargin (N). (O) Quantification of nuclear NFATc1 levels upon stimulation of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 196; n (NS) = 195. (P) Relative IL- 2 mRNA expression levels of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 7. (Q) Histograms and (R) quantification of up-regulated CD69 expression of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) after α-CD3 stimulation, n = 6–7. (B, D, E, F, H, J, K, M) Statistics: t test (B, D, F, H, J, 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: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

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

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative I/V relationships of TRPM7 channels in naïve CD4 T cells obtained via whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (B) Representative single-cell traces of cytosolic Ca 2+ concentrations of naïve CD4 T cells following α-CD3/α-CD28 stimulation. Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black) in saline (2 mM CaCl 2 ). (C, D, E, F) Quantification of Ca 2+ signals of naïve CD4 T cells for (C) basal, (D) delta Ca 2+ , (E) AUC and (F) oscillation frequency, n = 39–48 cells. (G) Representative I/V relationships of TRPM7 channels in total CD4 T cells obtained via whole-cell patch clamp using Mg 2+ -free intracellular solution. Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (H) Representative single-cell traces of cytosolic Ca 2+ concentrations of total CD4 T cells following α-CD3/α-CD28 stimulation. Data obtained as in (B). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black). (I, J, K, L) Quantification of Ca 2+ signals of total CD4 T cells for (I) basal, (J) delta Ca 2+ , (K) AUC, and (L) oscillation frequency, n = 29–30 cells. (M) Representative immune-fluorescence images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profiles of subcellular NFATc1 distribution (Ctrl, black; NS, red; DAPI, light blue) of naïve CD4 T cells treated with 30 μM NS8593 (NS) or DMSO (Ctrl) upon 30 min stimulation with α-CD3/α-CD28, scale bar = 2 μm. (N) Quantification of nuclear NFATc1 levels upon 30 min α-CD3/α-CD28 stimulation of cells treated with 30 μM NS8593 (NS, red) or DMSO (Ctrl, black), n (Ctrl) = 149; n (NS) = 144. (O) Representative immune-fluorescence images of NFATc1 localization (NFATc1 in red, DAPI in blue) and intensity profiles of subcellular NFATc1 distribution (Ctrl, black; NS, red; DAPI, light blue) of total CD4 T cells treated with 30 μM NS8593 (NS) or DMSO (Ctrl) upon 30 min stimulation with α-CD3/α-CD28, scale bar = 2 μm. (P) Quantification of nuclear NFATc1 levels upon 30 min α-CD3/α-CD28 stimulation in presence of 30 μM NS8593 (NS, red) cells or DMSO control (Ctrl, black) or, n (Ctrl) = 155; n (NS) = 132. (C, D, E, F, I, J, K, L, N, P) Statistics: t test (C, D, E, F, I, J, K, L, N, P). * P < 0.05; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Patch Clamp, Control, Saline, Fluorescence

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) IL-2 quantification in supernatant of naïve CD4 T cells 48 h after α-CD3/α-CD28 stimulation, n = 4–5. (B, C, D, E) Histograms and quantification of up-regulated activation markers CD69 (B, C) and CD25 (D, E) in naïve CD4 T lymphocytes 48 h after stimulation. Cells were treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (F) IL-2 quantification in supernatant of total CD4 T cells 48 h after α-CD3/α-CD28 stimulation or cells treated with 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 , n = 4–5. (G, H, I, J) Histograms and quantification of up-regulated activation markers CD69 (G, H) and CD25 (I, J) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with either 30 μM NS8593 or DMSO control, both with (Ctrl, blue; NS, orange) and without (Ctrl, black; NS, red) supplementation of 6 mM MgCl 2 . (K) Representative TRPM7 I/V relationships of total CD4 T cells obtained via whole-cell patch clamp with Mg 2+ -free intracellular solution. Cells were treated with 10 μM Waixenicin A (WxA, green) or EtOH control (Ctrl, black). (L, M, N, O) Histograms and quantification of up-regulated activation markers CD69 (L, M) and CD25 (N, O) in total CD4 T lymphocytes 48 h after stimulation. Cells treated with 10 μM Waixenicin A or EtOH control, both with (Ctrl, blue; WxA, light green) and without (Ctrl, black; WxA, green) supplementation of 6 mM MgCl 2 , n = 7. (P) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various NS8593 concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (Q). (Q) Respective quantification of NS8593 dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (P), n = 4–7. (R) Representative histograms of dose-dependent proliferation (CSFE dye dilution) of total CD4 T cells in presence of various Waixenicin A concentrations, with (right) and without (left) supplementation of 6 mM MgCl 2 . Cells gated on T cell population, single cells and CD4 + T cells. Color code as in (S). (S) Respective quantification of Waixenicin A dose-dependent proliferation of total CD4 T cells, with and without supplementation of 6 mM MgCl 2 , corresponding to (S), n = 4–8. (A, C, E, F, H, J, M, O, Q, S) Statistics: one-way ANOVA (A, C, E, F, H, J, M, O, Q, S). * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Activation Assay, Control, Patch Clamp

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative Western blot of pAKT Ser473 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO (Ctrl) in resting state and after 30 min α-CD3/α-CD28 stimulation. (B) Respective quantification of pAKT Ser473 signals from blots shown in (A), n = 6. (C) Representative immune-fluorescence images of AKT1-TRPM7 proximity-ligation interaction in CD4 T cells in resting state or after stimulation with α-CD3/α-CD28 for 30 min. AKT1-TRPM7 interactions are visible as red dots, DAPI in blue, scale bar = 5 μm. (D) Respective quantification of PLA signals per cell upon α-CD3/α-CD28 stimulation as shown in (C). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 173; n (NS) = 115. (E) Representative Western blot of pSMAD2 Ser465/Ser467 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min TGF-β stimulation. (F) Respective quantification of pSMAD2 Ser465/Ser467 signals from blots shown in E, n = 6. (G) Histogram and (H) quantification of pSMAD2 Ser465/Ser467 signals in CD4 T cells after 15 min TGF-β stimulation. Cells treated with 30 μM NS8593 (NS) or DMSO control (Ctrl), unstimulated Ctrl shown in light gray, n = 6. (I) Representative Western blot of pSMAD2 Ser465/Ser467 signals in CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 ) or H 2 O control (Ctrl) in resting state and after 10 min TGF-β stimulation. (J) Respective quantification of pSMAD2 Ser465/Ser467 signals from blots shown in (I), n = 4. (K) Histogram and (L) quantification of pSMAD2 Ser465/Ser467 signals in CD4 T cells after 15 min TGF-β stimulation. Cells treated with 6 mM MgCl 2 (MgCl 2 , blue) or H 2 O control (Ctrl, black), unstimulated Ctrl shown in light gray, n = 6–7. (M) Representative immune-fluorescence images of SMAD2-TRPM7 proximity-ligation interaction in CD4 T cells in resting state or after stimulation with TGF-β for 10 min. SMAD2-TRPM7 interactions are visible as red dots, DAPI in blue, scale bar = 5 μm. (N) Respective quantification of PLA signals per cell upon TGF-β stimulation as shown in (M). Cells were treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n (Ctrl) = 327; n (NS) = 284. (B, D, F, H, J, L, N) Statistics: one-way ANOVA (B, F, J) and t test (D, F, H, L, N). * P < 0.05; ** P < 0.005; **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Western Blot, Fluorescence, Ligation, Control

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Schematic description of naïve CD4 T-cell differentiation towards FOXP3-expressing regulatory T cells and RORƔt-expressing T H 17 cells, including respective cytokine polarization milieus. (B, C) Percentages of CD45RA − cells and (C) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6–7. (D, E) Representative FACS histograms and (E) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 6–7. (F, G) Percentages of CD45RA − cells and (G) CD25 + CD127 lo cells upon polarization of naïve CD4 T cells toward iT reg cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 7. (H, I) Representative FACS histograms and (I) quantification of FOXP3 expression levels of CD25 + CD127 lo iT reg cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (J) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of various NS8593 concentrations (red) compared with DMSO control (Ctrl, black), n = 6. (K, L) Representative FACS histograms and (L) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of various NS8593 concentrations (red) or DMSO control (Ctrl, black), n = 4–6. (M) Percentages of CCR6 + cells upon polarization of naïve CD4 T cells towards iT H 17 cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 6. (N, O) Representative FACS histograms and (O) quantification of RORƔt expression levels of CCR6 + iT H 17 cells upon 6 d polarization of naïve CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 , blue) compared with H 2 O control (Ctrl, black), n = 5. (P) Graphical summary of TRPM7-(in)dependent T-cell activation and differentiation towards iT reg and iT H 17 cells. Pharmacological blockade of TRPM7 reduces intracellular Mg 2+ levels, leads to reduced Ca 2+ signaling and results in reduced IL-2 secretion, impaired up-regulation of T-cell activation markers CD69 and CD25, and diminished proliferation upon TCR stimulus (left). TRPM7 inhibition during polarization of naïve CD4 T cells into iT reg cells preserves FOXP3 + signals of CD25 + CD127 lo iT reg cells. Polarization of naïve CD4 T cells into iT H 17 cells results in augmented RORƔt expression in the presence of 6 mM Mg 2+ , which is reduced upon TRPM7 inhibition, highlighting the need for Mg 2+ uptake and related TRPM7-dependent intracellular signaling for iT H 17 cell polarization (right). (B, C, E, F, G, I, J, L, M, O) Statistics: one-way ANOVA (B, C, E, J, L) and t test (F, G, I, 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: Cells were stained with primary antibodies overnight at 4°C, αTRPM7 (ACC-047; Alomone or MA527620 ; Thermo Fisher Scientific) or α-AKT1 (D9-9-C9; Thermo Fisher Scientific) or α-SMAD2 (sc-101153; Santa Cruz).

Techniques: Cell Differentiation, Expressing, Control, Activation Assay, Inhibition