Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

Techniques: Flow Cytometry, Purification, Activation Assay, Staining

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

Techniques: Incubation

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

Article Snippet: Permeabilized cells were incubated overnight with anti-Hvcn1 Ab (Alomone, catalog AHC-001).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

Techniques: Flow Cytometry, Purification, Activation Assay, Staining

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

Techniques: Incubation

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.147814

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

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

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

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: 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 .

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, Amplification, Western Blot, Concentration Assay

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: 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: Western Blot, Infection, Lysis, Isolation, shRNA, Transduction, Expressing, Two Tailed Test

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: 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 ). 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 .

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, Binding Assay, Staining, Transduction, Purification, SDS Page, Western Blot, Isolation

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data . 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

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

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: 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 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 and .

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: Western Blot, Expressing, Luciferase, Migration, Molecular Weight, Generated, Infection, Two Tailed Test

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: 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: Isolation, Infection, Two Tailed Test

Journal: Nature microbiology

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

doi: 10.1038/s41564-021-00890-3

Figure Lengend Snippet: A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell 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 ( – ). 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 . 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 .

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

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Hv1 is functionally expressed in spinal microglia and upregulated after SNT. A Representative images of an acute spinal cord slice with a patched GFP-expressing microglia (boxed area was enlarged, upper). Scale bar: 200 µm (left), 40 µm (right). Outward proton currents induced by voltage steps in spinal cord microglia (lower). Increased pH i decreased the outward proton current and shifted the activation threshold to more depolarized potentials in WT microglia. No outward current was observed in Hv1 KO microglia. B I/V curves showing that Hv1 KO abolished voltage-gated proton currents in spinal cord microglia (n = 6 cells from 3 mice each group). C, D Representative Western blot images ( C ) and quantification data ( D ) showing that Hv1 expression in the L4-5 level of the dorsal horn increased at POD1-7 after SNT. Data are presented as mean ± SEM, n = 4–5 mice/group. **p < 0.01, ***p < 0.001, GAPDH was used as internal control. Spinal cord tissue from Hv1 KO animals was used as a negative control to confirm the antibody specificity, one-way ANOVA with multi comparisons

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Western Blot, Negative Control

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Hv1 is required for pain hypersensitivity after SNT. A, B WT and Hv1 KO animals exhibit similar acute pain phenotypes in a tail flick test (A) and motor learning capabilities in a rotarod test ( B ). Data are presented as mean ± SEM, n = 5—6 for each group. WT vs. Hv1 KO., unpaired t-test for tail flick, one-way ANOVA for rotarod. C, D Hv1 KO mice display reduced mechanical allodynia ( C ) and thermal hyperalgesia (D) following SNT. Data are presented as mean ± SEM, n = 14 mice for each group. ** p < 0.01. WT ipsi vs. Hv1 KO ipsi., unpaired t-test. E, F Hv1 KO animals show reduced mechanical allodynia ( E ) and thermal hyperalgesia ( F ) following CPN ligation. Data are presented as mean ± SEM, n = 8 for WT, n = 7 for Hv1 KO. ** p < 0.01; *** p < 0.001. WT ipsi vs. Hv1 KO ipsi., unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Tail Flick Test, Ligation

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Hv1 deficiency does not alter microglial activation after SNT. A Representative images of Iba1 (top panels), BrdU (middle panels) and merged (bottom panels) immunoreactivity in the ipsilateral dorsal horn in control (left), SNT-induced WT (center) and SNT-induced Hv1 KO (right) mice at POD7. Inserts are higher magnification of boxed regions in the representative low magnification images. B Representative images of Iba1 (top panels), phosphorylated p38 (p-p38, middle panels) and merged (bottom panels) immunoreactivity in the ipsilateral dorsal horn in control (left), SNT-induced WT (center) and SNT-induced Hv1 KO (right) mice at POD3. Inserts are higher magnification of boxed regions. Scale bar:100 µm. C Quantitative summary of microglial density in the contralateral and ipsilateral dorsal horn as well as BrdU + microglia density in the ipsilateral dorsal horn at POD7 following SNT in WT and Hv1 KO mice (n = 10 slices from 3 mice for each group, unpaired t-test). D Quantitative p-p38 immunoreactivity in the contralateral and ipsilateral dorsal horn of WT and Hv1 KO mice. Data are presented as mean ± SEM, cells were randomly picked from 6 slices for each group. WT vs. Hv1 KO., unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Activation Assay

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: SNT-induced ROS production is attenuated in Hv1 KO mice. A Representative images of 8-OHG (top) and merged 8-OHG/Iba1 (bottom) immunostaining in WT (left) and Hv1 KO (right) spinal cord at POD7 after SNT. B Representative images of ox-DHE (top) and merged ox-DHE/CX 3 CR1-GFP (bottom) signals in WT (left) and Hv1 KO (right) spinal cord at POD3 after SNT. C Representative images of Gp91 phox (top) and merged Gp91 phox /Iba1 (bottom) immunostaining in WT (left) and Hv1KO (right) spinal cord at POD1 after SNT. Scale bar: 50 µm. D–F Quantitative data showing 8-OHG, ox-DHE and Gp91 phox signal in the ipsilateral dorsal horn after SNT. Data are presented as mean ± SEM, 7–15 slices from 3 mice for each group. * p < 0.05; ** p < 0.01; *** p < 0.001. WT vs. Hv1 KO unpaired t-test. G Mechanical allodynia in WT and Hv1 KO mice treated with and without ROS scavenger, sulforaphane (SF), 1 h after SNT (arrow). SF treatment significantly attenuated mechanical allodynia in WT mice when compared to control group (WT + saline) but not in H1KO mice with SF or saline treatment. Data are presented as mean ± SEM, n = 7 mice for each group. ** p < 0.01; *** p < 0.001, unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Immunostaining

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Reduced astrocyte activation in Hv1 KO spinal cord after SNT. A Representative images of Iba1 (top panels), GFAP (middle panels) and merged (bottom panels) immunoreactivity in dorsal horn of control (the contra side of a WT sample was shown, left), the ipsilateral of SNT-induced WT (left center), SNT-induced Hv1 KO (right center), and sulforaphane-treated SNT-induced WT (right) mice at POD 7. Inserts are higher magnification of boxed regions. Scale bar: 100 µm. B–D Sample dorsal horn slice ( B ) is labelled with GFAP-astrocytes (green) and Iba1-microglia (red) highlighting the different lamina of the spinal cord. Astrocytes ( C ) and microglia ( D ) density were quantified in WT and Hv1 KO tissues at POD 7. Scale bar: 100 µm. Data are presented as mean ± SEM, images obtained from 3 mice for each group. ** p < 0.01; *** p < 0.001, unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Activation Assay

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Hv1 is required for astrocyte-derived IFN-γ to promote neuropathic pain after SNT. A Real-time PCR analysis data showing mRNA expression fold change of glial-derived cytokines in the spinal cord at POD3 after SNT. Data are presented as mean ± SEM, n = 6 runs from 3 sample pools, each sample pool contained 2 mouse samples for each group . * p < 0.05 unpaired t-test. B Representative images of IFN-γ (top panels) and IFN-γ / GFAP merged (bottom panels) immunoreactivity in the ipsilateral dorsal horn in control (a naïve WT sample was shown, left), POD3 SNT-induced WT (left center), and Hv1 KO (center), as well as POD7 SNT-induced WT (right center) and Hv1 KO (right) tissues. Inserts are higher magnification of boxed regions. Scale bar: 50 µm. C Quantitation of IFN-γ immunoreactivity in control as well as POD3 and 7 SNT-treated WT and Hv1 KO mice. Data are presented as mean ± SEM, cells were randomly picked from 3 mice for each group. *** p < 0.001. WT vs. Hv1 KO, unpaired t-test. D Mechanical allodynia in WT and Hv1 KO mice treated with or without neutralizing IFN-γ antibody (anti-IFN-γ). Anti-IFN-γ (1 μg in 5 μl ACSF) was applied intrathecally at POD3 and POD4 after SNT. Anti-IFN-γ intrathecal injection attenuated mechanical allodynia in WT mice when compared to the control group (WT + saline) but not in HV1 KO mice with anti-IFN-γ or saline injection. Data are presented as mean ± SEM. n = 7 mice for each group, ** p < 0.01, unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay, Injection

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Hv1 is required for IFN-γ promotes neuropathic pain and astrocyte activation. A Mechanical allodynia in SNT mice treated with exogenous IFN-γ. IFN-γ (100U in 5 μl ACSF) was intrathecally applied at POD3 after SNT. Data are presented as mean ± SEM, n = 7 for control groups, n = 6 for IFN-γ treated group. # p < 0.05, Hv1 KO + saline vs Hv1 KO + IFN-γ, * p < 0.05, WT + saline vs. Hv1 KO + IFN-γ, unpaired t-test. B Mechanical allodynia in naïve mice treated with exogenous IFN-γ. IFN-γ (100U in 5 μl ACSF) was intrathecally applied after the baseline pain test. Data are presented as mean ± SEM, n = 6 mice for each group. ** p < 0.01, *** p < 0.001. WT vs. Hv1 KO, unpaired t-test. C Representative images of Iba1 (top panels), GFAP (middle panels) and merged (bottom panels) immunoreactivity in the dorsal horn in control (a naïve WT sample was shown, left), IFN-γ–treated WT (center) IFN-γ–treated Hv1 KO (right) mice at day 4 post injection. Scale bar: 50 µm. D , E , Quantification of microglia ( D ) and astrocyte ( E ) density at POD4 in Hv1 WT and KO mice following IFN-γ treatment. Data are presented as mean ± SEM, n = 6 slices from 3 mice for each group. ** p < 0.01. WT vs. Hv1 KO, unpaired t-test

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Activation Assay, Injection

Journal: Molecular Brain

Article Title: The voltage-gated proton channel Hv1 promotes microglia-astrocyte communication and neuropathic pain after peripheral nerve injury

doi: 10.1186/s13041-021-00812-8

Figure Lengend Snippet: Schematic diagram showing Hv1-dependent microglia-astrocyte interaction in neuropathic pain. After peripheral nerve injury, spinal microglia are activated and accompanied with Hv1 proton channel dependent ROS production. ROS acts on both neurons and astrocytes, triggering IFN-γ release. IFN-γ in turn contributes to further microglia activation and microglia-astrocyte interaction to maintain pain hypersensitivity after SNT

Article Snippet: Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology).

Techniques: Activation Assay