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    • anti hvcn1  (Alomone Labs)
    93
    Alomone Labs anti hvcn1
    T cells were purified from spleen and lymph nodes (LN) of WT mice and stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL) with 20 U/mL IL-2 for the indicated number of days. Expression of <t>Hvcn1</t> gene and protein was measured by quantitative PCR (qPCR) and Western blot in CD4 + and CD8 + T cell subsets ( A and B , respectively). ( C ) LN T cells from WT and Hvcn1-deficient mice were stained with DAPI (blue), anti-CD3 (orange) and anti-Hvcn1 (red) Abs and visualized by confocal microscopy. Scale bar: 10 μm. ( D and E ) Total T cells and B cells were isolated from WT mice ( n = 3) and the protein extract resolved by SDS gel electrophoresis. NADPH oxidase levels were normalized to GAPDH levels. ( F and G ) Relative pH i of naive (n)CD4 + and nCD8 + ( n = 3, F ) and Ab-activated (day 4, G ) CD4 + and CD8 + ( n = 6) WT and Hvcn1-deficient (KO) T cells was calculated by staining with pHRodo. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005; 2-tailed Student’s t test.
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    T cells were purified from spleen and lymph nodes (LN) of WT mice and stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL) with 20 U/mL IL-2 for the indicated number of days. Expression of Hvcn1 gene and protein was measured by quantitative PCR (qPCR) and Western blot in CD4 + and CD8 + T cell subsets ( A and B , respectively). ( C ) LN T cells from WT and Hvcn1-deficient mice were stained with DAPI (blue), anti-CD3 (orange) and anti-Hvcn1 (red) Abs and visualized by confocal microscopy. Scale bar: 10 μm. ( D and E ) Total T cells and B cells were isolated from WT mice ( n = 3) and the protein extract resolved by SDS gel electrophoresis. NADPH oxidase levels were normalized to GAPDH levels. ( F and G ) Relative pH i of naive (n)CD4 + and nCD8 + ( n = 3, F ) and Ab-activated (day 4, G ) CD4 + and CD8 + ( n = 6) WT and Hvcn1-deficient (KO) T cells was calculated by staining with pHRodo. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005; 2-tailed Student’s t test.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: T cells were purified from spleen and lymph nodes (LN) of WT mice and stimulated with plate-bound anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL) with 20 U/mL IL-2 for the indicated number of days. Expression of Hvcn1 gene and protein was measured by quantitative PCR (qPCR) and Western blot in CD4 + and CD8 + T cell subsets ( A and B , respectively). ( C ) LN T cells from WT and Hvcn1-deficient mice were stained with DAPI (blue), anti-CD3 (orange) and anti-Hvcn1 (red) Abs and visualized by confocal microscopy. Scale bar: 10 μm. ( D and E ) Total T cells and B cells were isolated from WT mice ( n = 3) and the protein extract resolved by SDS gel electrophoresis. NADPH oxidase levels were normalized to GAPDH levels. ( F and G ) Relative pH i of naive (n)CD4 + and nCD8 + ( n = 3, F ) and Ab-activated (day 4, G ) CD4 + and CD8 + ( n = 6) WT and Hvcn1-deficient (KO) T cells was calculated by staining with pHRodo. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005; 2-tailed Student’s t test.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Purification, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Confocal Microscopy, Isolation, SDS-Gel, Electrophoresis

    ( A and B ) WT male-derived skin rejection by WT ( n = 20) and Hvcn1-deficient (KO, n = 13) female mice ± SD. ( C ) Percentage of tetramer-positive CD8 + T cells in female mice after male-skin rejection. ( D ) Survival of B6Kd skin in WT ( n = 8), Hvcn1-deficient (KO, n = 10) mice, and Hvcn1-deficient mice CD8 + T cell-depleted/repleted with 5 × 10 5 WT cells ( n = 10). ( E ) Cell Trace Violet-labeled naive CD4 + and CD8 + T cell proliferation. ( F ) T cell subsets after proliferation. Representative histograms and bar charts of mean data ( n = 3, n = 3) are displayed ± SD. ( G and H ) Cytokine production and ( I and J ) T-bet expression measured in T cells ± SD ( n = 4) with representative dot plots. ( K ) TUNEL assay of Balb/C-derived IFN-γ–activated ECs cocultured for 5 hours with WT or Hvcn1-deficient Ab-activated CD8 + T cells. Representative images and mean number of apoptotic endothelial cells (ECs) are shown ± SD ( n = 5). Scale bar: 20 μm. ( L ) In vivo killing of female (F) or male (M) WT splenocytes stained with high and low CFSE concentrations by WT or Hvcn1-deficient females. Representative plot and histogram of differentially labeled splenocytes and proportion of CFSE hi (♀) to CFSE lo (♂) cells calculated 1 day later ± SD. ( M – O ) Viability of WT or Hvcn1-deficient T cells. Representative dot plots (M) and bar charts of mean apoptotic (A), late-apoptotic (LA), and viable (V) T cell proportions ( N and O ) ± SD ( n = 6). ( P and Q ) Cell marker expression by Ab-stimulated CD4 + or CD8 + T cells. Results presented as bar charts ± SD ( n = 4), with representative histograms. A and D, log-rank (Mantel-Cox) test. B , C , and E – Q , 2-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: ( A and B ) WT male-derived skin rejection by WT ( n = 20) and Hvcn1-deficient (KO, n = 13) female mice ± SD. ( C ) Percentage of tetramer-positive CD8 + T cells in female mice after male-skin rejection. ( D ) Survival of B6Kd skin in WT ( n = 8), Hvcn1-deficient (KO, n = 10) mice, and Hvcn1-deficient mice CD8 + T cell-depleted/repleted with 5 × 10 5 WT cells ( n = 10). ( E ) Cell Trace Violet-labeled naive CD4 + and CD8 + T cell proliferation. ( F ) T cell subsets after proliferation. Representative histograms and bar charts of mean data ( n = 3, n = 3) are displayed ± SD. ( G and H ) Cytokine production and ( I and J ) T-bet expression measured in T cells ± SD ( n = 4) with representative dot plots. ( K ) TUNEL assay of Balb/C-derived IFN-γ–activated ECs cocultured for 5 hours with WT or Hvcn1-deficient Ab-activated CD8 + T cells. Representative images and mean number of apoptotic endothelial cells (ECs) are shown ± SD ( n = 5). Scale bar: 20 μm. ( L ) In vivo killing of female (F) or male (M) WT splenocytes stained with high and low CFSE concentrations by WT or Hvcn1-deficient females. Representative plot and histogram of differentially labeled splenocytes and proportion of CFSE hi (♀) to CFSE lo (♂) cells calculated 1 day later ± SD. ( M – O ) Viability of WT or Hvcn1-deficient T cells. Representative dot plots (M) and bar charts of mean apoptotic (A), late-apoptotic (LA), and viable (V) T cell proportions ( N and O ) ± SD ( n = 6). ( P and Q ) Cell marker expression by Ab-stimulated CD4 + or CD8 + T cells. Results presented as bar charts ± SD ( n = 4), with representative histograms. A and D, log-rank (Mantel-Cox) test. B , C , and E – Q , 2-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Derivative Assay, Labeling, Expressing, TUNEL Assay, In Vivo, Staining, Marker

    Phosphorylation of Zap70, AKT, and S6 was measured by flow cytometry in purified naive WT or Hvcn1-deficient CD4 + ( A – C ) and CD8 + ( D – F ) T cells after Ab activation for the indicated time. Results are presented as the mean MFI ± SD. ( n = 3 independent experiments.) ( G and H ) Production of superoxide was evaluated by staining nCD4 + and nCD8 with DHE before activating with anti-CD3/28 + 20 U/mL IL-2 at the indicated time points. Cells were analyzed by flow cytometry. Right-hand side panels show the mean MFI (level) of DHE production, while the left-hand side graph shows the mean percentage of T cells producing DHE (± SD, n = 4). ( I and J ) Purified naive WT or Hvcn1-deficient CD4 + or CD8 + T cells were stained with Cell Trace Violet and then activated in culture with or without the indicated supplements and with 20 U/mL IL-2. On day 4, cells were harvested and counted. The mitotic index was calculated as a function of the number of cells and the percentage of cells in each division, as assessed by flow cytometry. Data are presented as mean ± SD. Student’s 2-sided t test and 1-way ANOVA with Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.005.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: Phosphorylation of Zap70, AKT, and S6 was measured by flow cytometry in purified naive WT or Hvcn1-deficient CD4 + ( A – C ) and CD8 + ( D – F ) T cells after Ab activation for the indicated time. Results are presented as the mean MFI ± SD. ( n = 3 independent experiments.) ( G and H ) Production of superoxide was evaluated by staining nCD4 + and nCD8 with DHE before activating with anti-CD3/28 + 20 U/mL IL-2 at the indicated time points. Cells were analyzed by flow cytometry. Right-hand side panels show the mean MFI (level) of DHE production, while the left-hand side graph shows the mean percentage of T cells producing DHE (± SD, n = 4). ( I and J ) Purified naive WT or Hvcn1-deficient CD4 + or CD8 + T cells were stained with Cell Trace Violet and then activated in culture with or without the indicated supplements and with 20 U/mL IL-2. On day 4, cells were harvested and counted. The mitotic index was calculated as a function of the number of cells and the percentage of cells in each division, as assessed by flow cytometry. Data are presented as mean ± SD. Student’s 2-sided t test and 1-way ANOVA with Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.005.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Flow Cytometry, Purification, Activation Assay, Staining

    The ECAR (mPh/min) was measured in naive (gray) and 4-day activated (blue) Hvcn1-deficient (circles) and WT (squares) CD4 + ( A ) and CD8 + ( B ) T cells. The bar graph shows the mean peak ECAR measured in WT (gray bars) and Hvcn1-deficient (open bars) T cells (± SD; n = 10–12). ( C – F ) The OCR (pmol/min) was analyzed to evaluate OXPHOS: Naive, C and E , and activated, D and F , WT (squares) and Hvcn1-deficient (circles) CD4 + , C and D , and CD8 + , E and F , T cells were sequentially incubated in glucose containing media, with Oligomycin, FCCP and Antimycin plus Rotenone while the OCR was measured. The OCR was used to calculate basal and maximal respiration as well as ATP production of WT (dark gray bars) and Hvcn1-deficient (white bars) T cells (± SD; n = 10–12). Results are presented as mean ± SD ( n = 5); 1-way ANOVA with Tukey post hoc test; * P < 0.05, *** P < 0.005.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: The ECAR (mPh/min) was measured in naive (gray) and 4-day activated (blue) Hvcn1-deficient (circles) and WT (squares) CD4 + ( A ) and CD8 + ( B ) T cells. The bar graph shows the mean peak ECAR measured in WT (gray bars) and Hvcn1-deficient (open bars) T cells (± SD; n = 10–12). ( C – F ) The OCR (pmol/min) was analyzed to evaluate OXPHOS: Naive, C and E , and activated, D and F , WT (squares) and Hvcn1-deficient (circles) CD4 + , C and D , and CD8 + , E and F , T cells were sequentially incubated in glucose containing media, with Oligomycin, FCCP and Antimycin plus Rotenone while the OCR was measured. The OCR was used to calculate basal and maximal respiration as well as ATP production of WT (dark gray bars) and Hvcn1-deficient (white bars) T cells (± SD; n = 10–12). Results are presented as mean ± SD ( n = 5); 1-way ANOVA with Tukey post hoc test; * P < 0.05, *** P < 0.005.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Incubation

    ( A and B ) Purified naive CD8 + WT and Hvcn1-deficient T cells were Ab-activated for 4 days, then lysed and fractionated into Cytoplasmic/Membrane (C/Me), mitochondrial (M), and nuclear (N) and were then analyzed by Western blot for the presence of Hvcn1, Hsp60, and Gapdh proteins. ( C ) Naive WT and Hvcn1-deficient CD8 + T cells were incubated with or without FCCP for 5 minutes before being stained with DHE and analyzed by flow cytometry. The mean percentage of DHE + T cells is shown (± SD, n = 4). ( D and E ) Total DNA was isolated from naive and Ab-activated (4 days) WT and Hvcn1-deficient CD8 + T cells ( n = 3–4). Quantitative PCR was used to assess expression of mitochondrial genes 16s and Nd1 and normalized to the nuclear gene Hk2 to calculate mitochondrial DNA copy number. Data are presented as mean ± SEM ( n = 5). Student’s 2-tailed t test; * P < 0.05, *** P < 0.005. ( F ) MMP of naive WT and Hvcn1-deficient CD8 + T cells was determined using MitoTracker Red (MitoRed). A representative histogram is shown on the right-hand side. Bar charts show mean MFI ± SD ( n = 3). Data are presented as mean ± SD ( n = 5). Student’s 2-tailed t test; *** P < 0.005.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: ( A and B ) Purified naive CD8 + WT and Hvcn1-deficient T cells were Ab-activated for 4 days, then lysed and fractionated into Cytoplasmic/Membrane (C/Me), mitochondrial (M), and nuclear (N) and were then analyzed by Western blot for the presence of Hvcn1, Hsp60, and Gapdh proteins. ( C ) Naive WT and Hvcn1-deficient CD8 + T cells were incubated with or without FCCP for 5 minutes before being stained with DHE and analyzed by flow cytometry. The mean percentage of DHE + T cells is shown (± SD, n = 4). ( D and E ) Total DNA was isolated from naive and Ab-activated (4 days) WT and Hvcn1-deficient CD8 + T cells ( n = 3–4). Quantitative PCR was used to assess expression of mitochondrial genes 16s and Nd1 and normalized to the nuclear gene Hk2 to calculate mitochondrial DNA copy number. Data are presented as mean ± SEM ( n = 5). Student’s 2-tailed t test; * P < 0.05, *** P < 0.005. ( F ) MMP of naive WT and Hvcn1-deficient CD8 + T cells was determined using MitoTracker Red (MitoRed). A representative histogram is shown on the right-hand side. Bar charts show mean MFI ± SD ( n = 3). Data are presented as mean ± SD ( n = 5). Student’s 2-tailed t test; *** P < 0.005.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Purification, Western Blot, Incubation, Staining, Flow Cytometry, Isolation, Real-time Polymerase Chain Reaction, Expressing

    Purified naive and 48-hour activated WT and Hvcn1-deficient CD8 T cells were incubated with 13 C 6 -Glucose for 18 hours, followed by metabolite extraction for LC-MS/MS analysis. Columns 1 and 3 show total levels of each metabolite in the samples. Columns 2 and 4 show the proportion of isotopologues of each metabolite indicated by “M+ n ,” which designates the position in the molecule where the 13 C label is found. ( A – C ) Fractional enrichment of glycolysis ( A ), TCA cycle ( B ), and glutamine metabolism ( C ) related 13 C-isotopologues. Data are presented as mean ± SEM; 2-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001; or Mann-Whitney test. Source data for this figure are available as supplemental material (Supplemental Data).

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: Purified naive and 48-hour activated WT and Hvcn1-deficient CD8 T cells were incubated with 13 C 6 -Glucose for 18 hours, followed by metabolite extraction for LC-MS/MS analysis. Columns 1 and 3 show total levels of each metabolite in the samples. Columns 2 and 4 show the proportion of isotopologues of each metabolite indicated by “M+ n ,” which designates the position in the molecule where the 13 C label is found. ( A – C ) Fractional enrichment of glycolysis ( A ), TCA cycle ( B ), and glutamine metabolism ( C ) related 13 C-isotopologues. Data are presented as mean ± SEM; 2-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001; or Mann-Whitney test. Source data for this figure are available as supplemental material (Supplemental Data).

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

    ( A and B ) Purified naive or Ab-activated (4 days) CD8 + WT and Hvcn1-deficient T cells were lysed and analyzed by Western blotting for the presence of phosphorylated and total AMPK. Quantification (pAMPK/total AMPK) is shown in B ( n = 3). ( C and D ) Total DNA was isolated from Ab-activated (4 days) WT and Hvcn1-deficient CD8 + T cells ( n = 3–4) cultured in the presence of the AMPK inhibitor SBI or vehicle alone. Quantitative PCR was used to assess expression of mitochondrial genes 16s in C and Nd1 in D and normalized to the nuclear gene Hk2 to calculate mitochondrial DNA copy number. Data are presented as mean ± SEM ( n > 3). Student’s 2-tailed t test; * P < 0.05, ** P < 0.01.

    Journal: JCI Insight

    Article Title: Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

    doi: 10.1172/jci.insight.147814

    Figure Lengend Snippet: ( A and B ) Purified naive or Ab-activated (4 days) CD8 + WT and Hvcn1-deficient T cells were lysed and analyzed by Western blotting for the presence of phosphorylated and total AMPK. Quantification (pAMPK/total AMPK) is shown in B ( n = 3). ( C and D ) Total DNA was isolated from Ab-activated (4 days) WT and Hvcn1-deficient CD8 + T cells ( n = 3–4) cultured in the presence of the AMPK inhibitor SBI or vehicle alone. Quantitative PCR was used to assess expression of mitochondrial genes 16s in C and Nd1 in D and normalized to the nuclear gene Hk2 to calculate mitochondrial DNA copy number. Data are presented as mean ± SEM ( n > 3). Student’s 2-tailed t test; * P < 0.05, ** P < 0.01.

    Article Snippet: After Fix/Perm cells were stained with anti–IFN-γ–APC (eBioscience, catalog XMG1.2), anti–IL-2–af488 (eBioscience, catalog JES6-5H4), anti–Granzyme B–FITC-CELL (BioLegend, catalog GB11), anti–Hvcn1 (Alomone, catalog AHC-001), anti–T-bet–BV421 (BioLegend, catalog 4B10), anti–IL-17–ef450 (eBioscience, catalog eBio17B7), and anti–FoxP3-APC (eBioscience, catalog FJK-16s).

    Techniques: Purification, Western Blot, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Expressing