anti hvcn1 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti hvcn1 antibody
    <t>Hvcn1</t> is expressed by mitochondria and regulates mitochondrial electron transport chain. ( A and B ) Purified naive CD8 + WT and Hvcn1-deficient T cells were <t>Ab-activated</t> 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 <t>Ab-activated</t> (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
    Anti Hvcn1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells"

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.147814

    Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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
    Figure Legend Snippet: Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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

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

    Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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
    Figure Legend Snippet: Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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

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

    Functional features of Hvcn1-deficient T cells. ( 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
    Figure Legend Snippet: Functional features of Hvcn1-deficient T cells. ( 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

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

    Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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
    Figure Legend Snippet: Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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

    Techniques Used: Flow Cytometry, Purification, Activation Assay, Staining

    Altered metabolic responses by Hvcn1-deficient naive T cells. 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
    Figure Legend Snippet: Altered metabolic responses by Hvcn1-deficient naive T cells. 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

    Techniques Used: Incubation

    Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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
    Figure Legend Snippet: Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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

    Techniques Used: Purification, Incubation, Liquid Chromatography with Mass Spectroscopy

    The proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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
    Figure Legend Snippet: The proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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

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

    2) Product Images from "Genetic variation of staphylococcal LukAB toxin determines receptor tropism"

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    Journal: Nature microbiology

    doi: 10.1038/s41564-021-00890-3

    Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .
    Figure Legend Snippet: Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .

    Techniques Used: Binding Assay, Expressing, Luciferase, Staining, Transduction, Purification, SDS Page, Isolation

    CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.
    Figure Legend Snippet: CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.

    Techniques Used: Polymerase Chain Reaction, Infection, Lysis, Isolation, shRNA, Transduction, Expressing, Two Tailed Test

    Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).
    Figure Legend Snippet: Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).

    Techniques Used: Flow Cytometry, shRNA, Expressing, Binding Assay, Staining

    Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34
    Figure Legend Snippet: Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Generated

    Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .
    Figure Legend Snippet: Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .

    Techniques Used: shRNA, Selection, Transduction, Expressing, Polymerase Chain Reaction, Amplification, Concentration Assay

    Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.
    Figure Legend Snippet: Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.

    Techniques Used: Genotyping Assay, Isolation, Mouse Assay, Infection, Two Tailed Test

    Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).
    Figure Legend Snippet: Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).

    Techniques Used: Flow Cytometry, Expressing

    LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .
    Figure Legend Snippet: LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .

    Techniques Used: Expressing, Luciferase, Migration, Molecular Weight, Generated, Mouse Assay, Infection, Two Tailed Test

    3) Product Images from "Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells"

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.147814

    Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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
    Figure Legend Snippet: Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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

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

    Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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
    Figure Legend Snippet: Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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

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

    Functional features of Hvcn1-deficient T cells. ( 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
    Figure Legend Snippet: Functional features of Hvcn1-deficient T cells. ( 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

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

    Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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
    Figure Legend Snippet: Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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

    Techniques Used: Flow Cytometry, Purification, Activation Assay, Staining

    Altered metabolic responses by Hvcn1-deficient naive T cells. 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
    Figure Legend Snippet: Altered metabolic responses by Hvcn1-deficient naive T cells. 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

    Techniques Used: Incubation

    Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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
    Figure Legend Snippet: Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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

    Techniques Used: Purification, Incubation, Liquid Chromatography with Mass Spectroscopy

    The proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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
    Figure Legend Snippet: The proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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

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

    4) Product Images from "Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury, et al. Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury"

    Article Title: Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury, et al. Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury

    Journal: Glia

    doi: 10.1002/glia.23926

    Brain acidosis persisted for weeks after TBI and was associated with dysregulation of microglial‐specific Hv1 proton channel and NOX expression. The long‐term duration of brain acidosis following TBI was examined. (a) Extracellular brain pH (pH e ) was measured in sham mice and Day 14 and 28 after TBI ( n = 7, 6, and 6/group). (b) Extracellular brain lactate concentrations are shown for each time point ( n = 7, 6, and 6/group). (c) Extracellular reactive oxygen species (ROS e ) levels in the cortex were increased relative to sham control ( n = 5–6/group). Quantitative PCR analysis of gene expression of NOX1 and NOX2 in the cortex (d) and hippocampus (e) is shown. Gene expression of Hvcn1 (Hv1) was chronically upregulated in the cortex and hippocampus relative to sham control (f). Western blot analysis of protein expression for Hv1 in the cortex and hippocampus (g) and the representative blot image is shown (h). PLX5622‐treated mice showed a reduction in Hv1 gene expression relative to vehicle controls (i). Gene expression was normalized by GAPDH and expressed as a fold‐change relative to sham or vehicle control. Protein expression was normalized to β‐actin and expressed as a fold‐change relative to sham control. For all gene and protein expression experiments, n = 5/group. Abbreviations: Abs absorbance, hippo hippocampus, veh, vehicle, μmol, micromole, μg, microgram. Data for (a–g) were analyzed by one‐way ANOVA using Dunnett's multiple comparison test to determine differences between sham and each time point postinjury (* p
    Figure Legend Snippet: Brain acidosis persisted for weeks after TBI and was associated with dysregulation of microglial‐specific Hv1 proton channel and NOX expression. The long‐term duration of brain acidosis following TBI was examined. (a) Extracellular brain pH (pH e ) was measured in sham mice and Day 14 and 28 after TBI ( n = 7, 6, and 6/group). (b) Extracellular brain lactate concentrations are shown for each time point ( n = 7, 6, and 6/group). (c) Extracellular reactive oxygen species (ROS e ) levels in the cortex were increased relative to sham control ( n = 5–6/group). Quantitative PCR analysis of gene expression of NOX1 and NOX2 in the cortex (d) and hippocampus (e) is shown. Gene expression of Hvcn1 (Hv1) was chronically upregulated in the cortex and hippocampus relative to sham control (f). Western blot analysis of protein expression for Hv1 in the cortex and hippocampus (g) and the representative blot image is shown (h). PLX5622‐treated mice showed a reduction in Hv1 gene expression relative to vehicle controls (i). Gene expression was normalized by GAPDH and expressed as a fold‐change relative to sham or vehicle control. Protein expression was normalized to β‐actin and expressed as a fold‐change relative to sham control. For all gene and protein expression experiments, n = 5/group. Abbreviations: Abs absorbance, hippo hippocampus, veh, vehicle, μmol, micromole, μg, microgram. Data for (a–g) were analyzed by one‐way ANOVA using Dunnett's multiple comparison test to determine differences between sham and each time point postinjury (* p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot

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    Alomone Labs anti hvcn1 antibody
    <t>Hvcn1</t> is expressed by mitochondria and regulates mitochondrial electron transport chain. ( A and B ) Purified naive CD8 + WT and Hvcn1-deficient T cells were <t>Ab-activated</t> 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 <t>Ab-activated</t> (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
    Anti Hvcn1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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

    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: Hvcn1 is expressed by mitochondria and regulates mitochondrial electron transport chain. ( 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

    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

    Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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

    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: Activation of AMPK maintains mitochondrial mass in Hvcn1-deficient CD8 + T cell activation. ( 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

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

    Functional features of Hvcn1-deficient T cells. ( 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

    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: Functional features of Hvcn1-deficient T cells. ( 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

    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: Functional Assay, Derivative Assay, Mouse Assay, Labeling, Expressing, TUNEL Assay, In Vivo, Staining, Marker

    Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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

    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: Altered TCR signaling and ROS production by Hvcn1-deficient T cells. 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

    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

    Altered metabolic responses by Hvcn1-deficient naive T cells. 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

    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: Altered metabolic responses by Hvcn1-deficient naive T cells. 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

    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

    Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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

    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: Metabolic analysis of Hvcn1-deficient CD8 + T cells. 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

    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

    The proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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

    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 proton channel Hvcn1 is expressed by T lymphocytes and regulates intracellular acidity. 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

    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, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Confocal Microscopy, Isolation, SDS-Gel, Electrophoresis

    Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Binding Assay, Expressing, Luciferase, Staining, Transduction, Purification, SDS Page, Isolation

    CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Polymerase Chain Reaction, Infection, Lysis, Isolation, shRNA, Transduction, Expressing, Two Tailed Test

    Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Flow Cytometry, shRNA, Expressing, Binding Assay, Staining

    Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Expressing, Derivative Assay, RNA Sequencing Assay, Generated

    Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: shRNA, Selection, Transduction, Expressing, Polymerase Chain Reaction, Amplification, Concentration Assay

    Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Genotyping Assay, Isolation, Mouse Assay, Infection, Two Tailed Test

    Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Flow Cytometry, Expressing

    LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .

    Article Snippet: mouse anti-His tag (1:3000, CSI20563B, Cell Sciences), rabbit anti-HVCN1 (1:1000, OAPB01154, Aviva Systems Biology), rabbit anti-HVCN1 (1:200, AHC-001, Alomone Labs), rabbit anti-HVCN1 (1:1000, PA5–24964, Invitrogen, Thermo Fisher Scientific), mouse anti-β-Actin (1:1000, 8H10D10, Cell Signaling Technology), mouse PE/Cy7 anti-human CD19 (1:100, 302215, BioLegend), mouse FITC anti-human CD3 (1:100, 300406, BioLegend), mouse Alexa Fluor® 700 anti-human CD14 (1:100, 325614, BioLegend), mouse PE anti-human CD4 (1:100, 317410, BioLegend), mouse APC anti-human CD8a (1:50, 300912, BioLegend), mouse APC anti-human CD11b (1:100, 301310, BioLegend), mouse Alexa Fluor® 700 anti-mouse/human CD11b (1:300, 101222, BioLegend).

    Techniques: Expressing, Luciferase, Migration, Molecular Weight, Generated, Mouse Assay, Infection, Two Tailed Test

    Brain acidosis persisted for weeks after TBI and was associated with dysregulation of microglial‐specific Hv1 proton channel and NOX expression. The long‐term duration of brain acidosis following TBI was examined. (a) Extracellular brain pH (pH e ) was measured in sham mice and Day 14 and 28 after TBI ( n = 7, 6, and 6/group). (b) Extracellular brain lactate concentrations are shown for each time point ( n = 7, 6, and 6/group). (c) Extracellular reactive oxygen species (ROS e ) levels in the cortex were increased relative to sham control ( n = 5–6/group). Quantitative PCR analysis of gene expression of NOX1 and NOX2 in the cortex (d) and hippocampus (e) is shown. Gene expression of Hvcn1 (Hv1) was chronically upregulated in the cortex and hippocampus relative to sham control (f). Western blot analysis of protein expression for Hv1 in the cortex and hippocampus (g) and the representative blot image is shown (h). PLX5622‐treated mice showed a reduction in Hv1 gene expression relative to vehicle controls (i). Gene expression was normalized by GAPDH and expressed as a fold‐change relative to sham or vehicle control. Protein expression was normalized to β‐actin and expressed as a fold‐change relative to sham control. For all gene and protein expression experiments, n = 5/group. Abbreviations: Abs absorbance, hippo hippocampus, veh, vehicle, μmol, micromole, μg, microgram. Data for (a–g) were analyzed by one‐way ANOVA using Dunnett's multiple comparison test to determine differences between sham and each time point postinjury (* p

    Journal: Glia

    Article Title: Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury, et al. Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury

    doi: 10.1002/glia.23926

    Figure Lengend Snippet: Brain acidosis persisted for weeks after TBI and was associated with dysregulation of microglial‐specific Hv1 proton channel and NOX expression. The long‐term duration of brain acidosis following TBI was examined. (a) Extracellular brain pH (pH e ) was measured in sham mice and Day 14 and 28 after TBI ( n = 7, 6, and 6/group). (b) Extracellular brain lactate concentrations are shown for each time point ( n = 7, 6, and 6/group). (c) Extracellular reactive oxygen species (ROS e ) levels in the cortex were increased relative to sham control ( n = 5–6/group). Quantitative PCR analysis of gene expression of NOX1 and NOX2 in the cortex (d) and hippocampus (e) is shown. Gene expression of Hvcn1 (Hv1) was chronically upregulated in the cortex and hippocampus relative to sham control (f). Western blot analysis of protein expression for Hv1 in the cortex and hippocampus (g) and the representative blot image is shown (h). PLX5622‐treated mice showed a reduction in Hv1 gene expression relative to vehicle controls (i). Gene expression was normalized by GAPDH and expressed as a fold‐change relative to sham or vehicle control. Protein expression was normalized to β‐actin and expressed as a fold‐change relative to sham control. For all gene and protein expression experiments, n = 5/group. Abbreviations: Abs absorbance, hippo hippocampus, veh, vehicle, μmol, micromole, μg, microgram. Data for (a–g) were analyzed by one‐way ANOVA using Dunnett's multiple comparison test to determine differences between sham and each time point postinjury (* p

    Article Snippet: The membrane was incubated in rabbit anti‐HVCN1 (1:10000; Cat# AHC‐001, Alomone labs, Jerusalem, Israel), or mouse anti‐β‐actin (1:5000; Cat# A5441, Sigma‐Aldrich) overnight at 4°C, then washed 3 times in PBS‐T, and incubated in appropriate secondary antibodies for 2 hr at room temperature.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot