asic2  (Alomone Labs)

Journal: Scientific Reports

Article Title: The relationship between acid-sensing ion channel, ASIC2 , and oncogenic β-catenin signaling in ovarian cancer

doi: 10.1038/s41598-025-03429-2

Figure Lengend Snippet: ASIC2 correlation with PDE10. ( A ) Kaplan-Meier survival plot of PDE10 High and PDE10 Low mRNA expression in ovarian tumors from the TCGA. P-Value: 5.490e-3. ( B ) Expression of PDE10 mRNA in PDE10 High and PDE10 Low ovarian tumors. **** P < 0.0001. ( C ) ASIC2 expression in PDE10 High and PDE10 Low ovarian tumors. ** P < 0.01. ( D ) Western blot analysis of ASIC2 in ES2, SKOV3, and OVCAR8 cells. Quantification of ASIC2 is shown. β-ACTIN is used as a loading control. ASIC2 expression and localization in ovarian cancer cells. ASIC2 (Green, top left) or IgG control (top right) and merged with nuclei (Blue, bottom) for ( E ) ES2, ( F ) SKOV3, and ( G ) OVCAR8. Scale bar = 100 μm.

Article Snippet: Primary antibodies were used to detect ASIC2 (ThermoFisher #PA5-26222), non-phospho-β-catenin S45 (CellSignaling #19807, RRID: AB_2650576), non-phospho-β-catenin S33/37/T41 (CellSignaling #8814, RRID: AB_11127203), C-MYC (CellSignaling #5605, RRID: AB_1903938) IgG (abcam #ab171870, RRID: AB_2687657).

Techniques: Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: The relationship between acid-sensing ion channel, ASIC2 , and oncogenic β-catenin signaling in ovarian cancer

doi: 10.1038/s41598-025-03429-2

Figure Lengend Snippet: Growth inhibition using ASIC2 inhibitors. Amiloride and Diminazene were analyzed for their ability to inhibit growth at multiple concentrations in ( A ) ES2, ( B ) SKOV3, and ( C ) OVCAR8. Cells were treated for 24 h with Diminazene (10 µM) and Amiloride (25 µM) and probed for expression of PDE10 and ASIC2. ( D ) PDE10 and ASIC2 are decreased with both diminazene and amiloride in ES2 cells. ( E ) PDE10 and ASIC2 are decreased with diminazene and amiloride in SKOV3 cells. ( F ) ASIC2 but not PDE10 is decreased with diminazene and amiloride in OVCAR8 cells. Quantification of PDE10 and ASIC2 blots is shown in D – F .

Article Snippet: Primary antibodies were used to detect ASIC2 (ThermoFisher #PA5-26222), non-phospho-β-catenin S45 (CellSignaling #19807, RRID: AB_2650576), non-phospho-β-catenin S33/37/T41 (CellSignaling #8814, RRID: AB_11127203), C-MYC (CellSignaling #5605, RRID: AB_1903938) IgG (abcam #ab171870, RRID: AB_2687657).

Techniques: Inhibition, Expressing

Journal: Scientific Reports

Article Title: The relationship between acid-sensing ion channel, ASIC2 , and oncogenic β-catenin signaling in ovarian cancer

doi: 10.1038/s41598-025-03429-2

Figure Lengend Snippet: ASIC2 expression in PDE10 knockout cells. ( A ) Previously generated SKOV3 PDE10 knockout cells were analyzed for expression of ASIC2 by western blot. GAPDH serves as a loading control. Quantification of PDE10 and ASIC2 is shown. ( B ) ASIC2 expression was analyzed by confocal immunofluorescence and is shown on top in green and on bottom merged with nuclei in blue. Scale bar = 100 μm. ( C ) Quantitative nuclear fluorescence analysis of cells in B show a statistically significant reduced expression of ASIC2 in PDE10 knockout cells. ( D ) PDE10-KO cells were analyzed for survival after treatment with Diminazene for 72 h. IC50 values for each clone is shown.

Article Snippet: Primary antibodies were used to detect ASIC2 (ThermoFisher #PA5-26222), non-phospho-β-catenin S45 (CellSignaling #19807, RRID: AB_2650576), non-phospho-β-catenin S33/37/T41 (CellSignaling #8814, RRID: AB_11127203), C-MYC (CellSignaling #5605, RRID: AB_1903938) IgG (abcam #ab171870, RRID: AB_2687657).

Techniques: Expressing, Knock-Out, Generated, Western Blot, Control, Immunofluorescence, Fluorescence

Journal: Scientific Reports

Article Title: The relationship between acid-sensing ion channel, ASIC2 , and oncogenic β-catenin signaling in ovarian cancer

doi: 10.1038/s41598-025-03429-2

Figure Lengend Snippet: Downstream ASIC signaling in ovarian cancer. ( A ) SKOV3 PDE10-KO cells were probed for expression of C-MYC. ( B ) Quantification of nuclear C-MYC expression from A. ( C ) Promoter region of ASIC2 containing highlighted MYC binding sites. ( D ) Previously published consensus binding site of MYC . ( E ) Proposed signaling pathway describing the relationship between ASIC2 and PDE10 through the convergence on β-catenin and mediated by MYC. Scale bar = 100 μm.

Article Snippet: Primary antibodies were used to detect ASIC2 (ThermoFisher #PA5-26222), non-phospho-β-catenin S45 (CellSignaling #19807, RRID: AB_2650576), non-phospho-β-catenin S33/37/T41 (CellSignaling #8814, RRID: AB_11127203), C-MYC (CellSignaling #5605, RRID: AB_1903938) IgG (abcam #ab171870, RRID: AB_2687657).

Techniques: Expressing, Binding Assay

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Protocol of mouse tube test assay. Four mice housed together for 2 weeks undergo a round-robin tournament to determine social ranking. ( B ) Typical ranking of a group of one Asic2 −/− mouse versus three WT cagemates. ( C ) Average number of winning times in trials 1 and 7. ( D ) Comparison of winning times between WT and Asic2 −/− mice shows that Asic2 −/− mice have higher winning times. **** P < 0.0001, one-way ANOVA. n = 28 cages. ( E ) Typical ranking of three Asic2 −/− mice versus one WT cagemate. ( F ) Average number of winning times in trials 1 and 7. ( G ) Three Asic2 −/− mice exhibit higher winning times than one WT cagemate. **** P < 0.0001; ns, nonsignificant, one-way ANOVA. n = 7 cages. ( H ) Comparison of winning times between groups of one Asic2 −/− mouse versus three WT cagemates and one WT mouse versus three Asic2 −/− cagemates, using data from (B) to (G). *** P = 0.0007, two-tailed unpaired Student’s t test. ( I ) Comparison of winning times between groups of one Asic1a −/− mouse versus three WT cagemates, and one WT mouse versus three Asic1a −/− cagemates. ns, P = 0.8477, two-tailed unpaired Student’s t test. n = 7 cages. ( J and K ) Schematic of mouse warm spot test and correlation with tube test rank. P = 0.0118, two-sided Fisher’s exact test. n = 16 cages. ( L and M ) Schematic of mouse urine marking test and correlation with tube test rank. P = 0.0118, two-sided Fisher’s exact test. n = 16 cages.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Comparison, Two Tailed Test, Spot Test

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Recordings of acid-induced currents in the mPFC slice. ( B to D ) pH-dependent ASIC currents in WT (B) ( n = 5 to 13 cells per pH dose, from four mice), Asic1a −/− (C) ( n = 5 to 21 cells per pH dose, from four mice) and Asic2 −/− (D) neurons ( n = 5 to 12 cells per pH dose, from 4 mice). ( E ) Normalized ASIC currents in WT (black) and Asic2 −/− (red) neurons. ( F ) Current density at pH 5.0 in WT and Asic2 −/− neurons. ns, P = 0.2828. ( G ) Desensitization decay times (τ), ** P = 0.0058, n = 16 cells per four mice. ( H ) Normalized ASIC current per unit time. **** P < 0.0001, n = 15 cells per four mice. ( I ) Representative Western blot data illustrating the expression of ASIC1a and ASIC2 in WT mice with different rankings in the tube test. ( J ) Average expression data for ASIC1a and ASIC2. ns, P = 0.1592, ** P < 0.007. ( K and L ) Correlation curves depicting expressions of ASIC1a [Pearson correlation coefficient ( r ) = −0.23, P = 0.18, ns] and ASIC2 ( r = −0.80, ** P = 0.005) in relation to social rankings, n = 9 mice in each ranking group. ( M ) Representative ASIC currents in neurons from winner (α) and loser (δ) mice. ( N and O ) Average current density and τ of ASIC currents. ns, P = 0.4211; * P = 0.0113, n = 21 cells per four mice. ( P ) pH-dependent ASIC currents in WT mice were analyzed to determine pH 50 values, obtained by best-fitting with the Hill equation. ( Q ) Average pH 50 between neurons in α and δ mice. **** P < 0.0001, n = 13 to 15 cells per four mice.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Western Blot, Expressing, Patch Clamp

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Schematic showing the injection of AAV 2 -CMV-mASIC2 into the mPFC in Asic2 −/− mice. ( B ) Left, a representative image of the AAV 2 -mASIC2 injection site and neurons expressing mASIC2; middle-right, representative recording of acid-induced ASIC-like currents in the GFP positive and negative neurons. ( C ) The representative comparison of acid-induced currents among WT, Asic2 −/− (non-GFP cells), and AAV-mASIC2 neurons (GFP positive cells). ( D and E ) Comparison of desensitization decay times (D) and density (E) of the acid-induced currents in the neurons in (C). WT versus Asic2 −/− , ** P = 0.0012, Asic2 −/− versus AAV-mASIC2, ** P = 0.0082; AAV-mASIC2 versus AAV-sham, * P = 0.0129, ns, P = 0.6763, one-way ANOVA. n = 12 to 15 cells from four mice. ( F ) Schematic showing the tube test protocol before and after AAV-mediated expression of ASIC2 in the mPFC of Asic2 −/− mice. ( G ) Representative result of ranking in the tube test following the restoration of ASIC2 in mPFC neurons of Asic2 −/− mice. ( H ) Average winning times in the tube test after restoring ASIC2 in mPFC neurons of Asic2 −/− mice. ( I ) Comparison of winning times between the group with restored ASIC2 in Asic2 −/− mice and one of its WT subordinates. ** P = 0.0092; ns, P = 0.3652, two-tailed unpaired Student’s t test. n = 9 cages.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Injection, Expressing, Comparison, Two Tailed Test, Over Expression, Patch Clamp

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Recording site schematic in the mPFC. ( B to D ) PPR recorded with 50- to 500-ms interstimulus intervals. Left: Representative PPR traces at 50 ms in WT and Asic2 −/− mice before and after social rankings. Right: Average PPR plotted against intervals. (C) * P = 0.0433; (D) ** P = 0.0088, 0.0044, * P = 0.0481, n = 10 cells per four mice. ( E ) Representative AMPAR-EPSCs (−80 mV) and NMDAR-EPSCs (+60 mV) in WT and Asic2 −/− neurons. ( F ) Average AMPAR/NMDAR ratios before and after social rankings. Current amplitudes measured 70 ms after onset. ns, P = 0.6117; * P = 0.0470; *** P = 0.0006. n = 22 to 29 cells per five mice. ( G and H ) mEPSCs in the groups of WT versus WT, and WT versus Asic2 −/− mice after social rankings. Upper: Representative mEPSC traces. Lower: Cumulative distributions of mEPSC amplitudes and interevent intervals. (G) * P = 0.0342; ns, P = 0.4768; (H) * P = 0.0109, 0.0065, n = 18 cells per four mice. ( I ) AMPAR current rectification in WT and Asic2 −/− neurons before social rankings. Left: Current-voltage relationships of AMPARs. Inset: Representative AMPAR-EPSC traces at −80 mV and +60 mV. NMDARs blocked with 100 μM d -APV. Right: AMPAR current rectification index (−80 mV/+60 mV). ns, P = 0.5913. n = 18 cells per four mice. ( J and K ) AMPAR current rectification of WT versus WT, and WT versus Asic2 −/− neurons after social rankings. ns, P = 0.4467; *** P = 0.0003, n = 22 cells per four mice. ( L to N ) Time course of 50 μM NASPM effect on EPSCs in mPFC neurons of WT versus WT (M) and Asic2 −/− versus WT (N) mice groups before (L) and after (M and N) social rankings. NMDARs blocked with 100 μM d -APV. Right: NASPM-sensitive EPSC comparison. ns, P = 0.9864, P = 0.1059; **** P < 0.0001, n = 10 cells per four mice. All comparisons by two-tailed unpaired Student’s t test.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Comparison, Two Tailed Test, Patch Clamp

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Dendritic and spine morphology of mPFC neurons using Alexa 568 dye. ( B ) Sholl analysis for dendritic branches. Left: Parameters measured with Sholl shells from the cell body. Right: Nodes (blue dots) are branching points, and intersections (yellow dots) are where processes intersect Sholl shells. ( C ) Reconstructed mPFC neurons from WT winner and loser mice. ( D to G ) Sholl analysis of dendritic intersections (D), ** P = 0.0019; number of branches (E), * P = 0.0314; branch length (F), ns, P = 0.1465; and node numbers (G), ns, P = 0.0717. n = 6 to 8 slices per four mice. ( H ) Reconstructed mPFC neurons from Asic2 −/− winner and WT loser mice. ( I to L ) Sholl analysis: dendritic intersections (I) **** P < 0.0001; branches (J) ** P = 0.0042; branch length (K) * P = 0.0409; and nodes (L) * P = 0.0412. n = 6 to 8 slices per four mice. ( M ) Spine structures in mPFC neurons from WT winner and loser mice. ( N ) Spine density comparison: mature, **** P < 0.0001; immature, ** P = 0.001; total spines, ns, P = 0.7656. ( O ) Immature spine density (stubby, thin, and filopodia) in WT winner and loser mice. **** P < 0.0001; * P = 0.0253; ns, P = 0.9336. ( P ) Mature-to-immature spine density ratio in WT winner and loser mice, **** P < 0.0001. n = 27 to 37 neurons per four mice. ( Q ) Spine structures in mPFC neurons from Asic2 −/− winner and WT loser mice. ( R ) Spine density comparison: mature, **** P < 0.0001; immature, **** P < 0.0001; total spines, ** P = 0.0026. ( S ) Immature spine density in Asic2 −/− winner and WT loser mice, **** P < 0.0001; ns, P = 0.0624, 0.6637. ( T ) Mature-to-immature spine density ratio in Asic2 −/− winner and WT loser mice, **** P < 0.0001. n = 27 neurons per four mice. All comparisons by two-tailed unpaired Student’s t test.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Morphology, Analysis, Comparison, Two Tailed Test

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Heatmap of substrate phosphorylation levels for STK in Asic2 −/− brain samples versus WT controls. ( B ) Global phosphorylation level differences between Asic2 −/− and WT brain samples. *** P = 0.0001 by paired two-tailed Student’s t test. n = 86 peptides. ( C ) Reverse KRSA plots mapping peptides to upstream kinases in Asic2 −/− versus WT brains. ( D ) Quantification of PKA and AKT ( Z > 2) using histogram peacock plots. ( E ) Waterfall plot of differentially identified kinases in Asic2 −/− brain samples versus WT controls. Red dots indicate increased or decreased representation for each kinase. ( F ) piNET analysis identified downstream proteins activated by STK pathways, highlighting PKA (PRKACA) and AKT1 pathways. Selected kinases (red nodes) and downstream regulators (blue nodes) are connected by green edges. ( G ) Asic2 −/− molecular interaction network. Functionally enriched protein-protein interaction network of upstream kinase “hits” (red) and interpolated hidden nodes (yellow) identified by Kinograte R software. n = 5 mice per group. ( H ) mRNA expression of PKA (PRKACA) via RT-qPCR in Asic2 −/− versus WT mice mPFC. * P = 0.0217 by unpaired two-tailed Student’s t test. n = 14 mice per group. ( I ) Basal cAMP level measured by ELISA in Asic2 −/− versus WT mice mPFC. ** P = 0.0039 by unpaired two-tailed Student’s t test. n = 6 to 9 mice per group. ( J ) mRNA expression of BDNF via RT-qPCR in Asic2 −/− versus WT mice mPFC. ** P = 0.0062 by unpaired two-tailed Student’s t test. n = 12 mice per group.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Phospho-proteomics, Two Tailed Test, Analysis, Software, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Schematic showing the AAV 2 -hSyn-Cre injection into the mPFC of Asic2 f/f mice. ( B ) Representative immunofluorescence image depicting the AAV 2 -hSyn-Cre injection site. ( C ) Representative immunofluorescence image demonstrating colocalization of ASIC2 (red) and AAV 2 -hSyn-Cre/AAV 2 -hSyn-GFP (green). Please note the presence of colocalization indicated by arrows in the AAV 2 -hSyn-GFP control group (upper), whereas such colocalization is absent in the AAV 2 -hSyn-Cre group (lower). ( D ) Western blot results show ASIC2 protein levels in the AAV 2 -hSyn-Cre and AAV 2 -hSyn-GFP groups. * P = 0.0472. n = 6 mice. ( E ) Schematic showing procedures of the tube test, AAV-hSyn-Cre injection into the mPFC of Asic2 f/f mice, and the EPSC recordings. ( F ) Summarized results of ranking in the tube test before and after virus injections. ( G ) Average percentage of winning times in the tube test before and after AAV 2 -hsyn-Cre and AAV 2 -hSyn-GFP injections. *** P = 0.0007, n = 9 cages. ( H ) Representative mEPSC traces of Asic2 f/f mPFC neurons in AAV 2 -hsyn-Cre and AAV 2 -hSyn-GFP groups. ( I and J ) Cumulative distributions of mEPSC amplitudes.* P = 0.0088. ( K and L ) Cumulative distributions of mEPSC interevent intervals, * P = 0.0172. n = 10 cells from four mice per group. ( M ) Representative mIPSC traces of Asic2 f/f mPFC neurons in AAV 2 -hsyn-Cre and AAV 2 -hSyn-GFP groups. ( N and O ) Cumulative distributions of mIPSC amplitudes, ns, P = 0.8873. ( P and Q ) Cumulative distributions of mEPSC interevent intervals, * P = 0.0115. n = 9 cells from four mice per group. All comparisons by two-tailed unpaired Student’s t test.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Injection, Immunofluorescence, Control, Western Blot, Virus, Two Tailed Test, Patch Clamp

Journal: Science Advances

Article Title: Mice lacking acid-sensing ion channel 2 in the medial prefrontal cortex exhibit social dominance

doi: 10.1126/sciadv.adn7573

Figure Lengend Snippet: ( A ) Generation of ASIC2-cKO mice in excitatory neurons (ASIC2-Vglut1-cKO) by crossbreeding Asic2 f/f mice with Vglut1-ires2-Cre mice. ( B ) Immunofluorescence images showing the absence of ASIC2 expression (green) in mPFC excitatory neurons labeled with N -methyl d -aspartate receptor subtype 2B (NR2B) (red). Inset highlights the lack of ASIC2 and NR2B colocalization. ( C ) One ASIC2-Vglut1-cKO and three WT mice cohoused for 2 weeks, followed by the tube test tournament to establish social rankings. Data from six cages per group summarized. ( D ) Comparison of time to stable rankings between ASIC2-Vglut1-cKO and WT subordinate mice. *** P = 0.0002, n = 6 cages. ( E ) Schematic of AAV 2 -CaMKIIα-Cre injection into Asic2 f/f mouse mPFC neurons. ( F ) Representative image of AAV 2 -CaMKIIα-Cre injection site. ( G ) Immunofluorescence image demonstrating ASIC2 (green) and AAV 2 -CaMKIIα-Cre/mCherry (red) colocalization. Arrows indicate colocalization in AAV 2 -CaMKIIα-mCherry, but not AAV 2 -CaMKIIα-Cre group. ( H ) Whole-cell patch-clamp recording of acid-induced ASIC-like currents in AAV 2 -CaMKIIα-Cre (mCherry positive) mPFC neurons. ( I ) Representative traces of acid-induced currents in AAV 2 -CaMKIIα-Cre (red) and AAV 2 -CaMKIIα-mCherry (black) neurons. ( J ) Comparison of desensitization decay times of acid-induced currents in neurons from (I). Effective ASIC2 deletion in AAV 2 -CaMKIIα-Cre neurons shows prolonged desensitization times. ** P = 0.0082. n = 15 to 17 cells from four mice per group. ( K ) Schematic of tube test procedures following AAV 2 -CaMKIIα-Cre injection into Asic2 f/f mouse mPFC. ( L ) Summary of tube test rankings before and after AAV 2 -CaMKIIα-Cre and AAV 2 -CaMKIIα-mCherry injections. ( M ) Average percentage of winning times in the tube test before and after AAV 2 -CaMKIIα-Cre and AAV 2 -CaMKIIα-mCherry injections. *** P = 0.0007, n = 6 cages. All comparisons by two-tailed unpaired Student’s t test.

Article Snippet: Primary antibodies used in this study are anti-ASIC1a antibody (Alomone Labs, no. ASC-014), anti-ASIC2 antibody (Alomone Labs, no. ASC-012), GluA1 polyclonal antibody (Thermo Fisher Scientific, no. PA5-99527), NMDAR2B polyclonal antibody (Innovative Research, no. 71-8600), PKA alpha antibody (Thermo Fisher Scientific, no. MA5-37857), and β-actin antibody (Cell Signaling Technology, no. 4967).

Techniques: Immunofluorescence, Expressing, Labeling, Comparison, Injection, Patch Clamp, Two Tailed Test

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: TaqMan Gene Expression Assay Primer‐Probe Pairs for mouse ENaC Subunits.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Gene Expression, Amplification

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Primary antibodies, species, titer, and source.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques:

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: qPCR detection of α, β, γENaC, and ASIC1‐5 transcript expression in cultured bone marrow derived macrophages and freshly isolated monocytes from male and female mice. Macrophages were cultured in the presence of 20 ng/mL of CSF1. (a) PCR reactions from macrophages were separated on 3%–4% agarose gels to determine if amplicon of expected size was present (identified by arrowhead in samples with >1 product). 100 ng RNA template equivalent was used for all reactions except ASIC1, where 1000 ng was used. Three primer pair‐probe sets were tested on ASIC2 and ASIC1. The primer pair‐probe sets shown amplified a band at the expected size, in addition to 1–2 other bands. (b) Macrophage C th 's individual ENaC and ASIC transcripts from bone marrow derived macrophages 3 replicates in n = 2 trials. Thresholds at or near 35 cycles were consistently identified in all replicates for αENaC and ASIC3. Thresholds less than 39 cycles were identified for βENaC and ASIC2. γENaC amplified in only 1/6 and 4/6 replicates in male and female samples, respectively. (c) Bone marrow derived freshly isolated monocytes C th 's for individual ENaC and ASIC subunits using 75 ng RNA template equivalent. Samples from four animals were pooled. (d) Bone marrow derived freshly isolated monocyte Western blot detection of αENaC and βENaC. Samples were pooled from four animals. *Indicates statistical difference between male and female at p < 0.05 using an Uncorrected Fisher LSD Test.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Expressing, Cell Culture, Derivative Assay, Isolation, Amplification, Western Blot

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Loss of βENaC or ASIC2 inhibits bone marrow monocyte chemotactic migration. Chemotactic migration is attenuated in freshly isolated bone marrow monocytes from (a, b) βENaC hypomorph mice (βENaC m/m , 10–13 weeks of age) or (c, d) ASIC2 global knockout mice (ASIC2 −/− , 7–8 weeks of age). Monocytes were isolated in parallel from an age‐matched wildtype and modified animal within a sex then migrated overnight. Mice were used between 7 and 13 weeks of age. These findings suggest (1) migration responses in wildtype mice are greater in female versus males, and (2) βENaC and ASIC2 both contribute to migration, but differentially in the sexes. Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males. Normalized migration data are shown in Figure Representative images are shown in panels a and c and group data are shown in panels b and d. Data are mean ± SEM and represent seven FOVs from three insets ( n = 21) and were analyzed using two‐way ANOVA followed by Holm‐Sidak post hoc test. p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Migration, Isolation, Knock-Out, Modification

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Loss of ASIC2 plus βENaC on bone marrow monocyte chemotactic migration is not additive. Chemotactic migration in freshly isolated bone marrow from mice homozygous for ASIC2 global knockout and βENaC hypomorph alleles (ASIC2 −/− /βENaC m/m , 6–7 weeks). Monocytes were isolated in parallel from an age‐matched wildtype and modified animal within a sex then migrated overnight. Representative images of underside of migration membrane (a) and group data (b) in males are shown. These findings suggest (1) migration responses in wildtype mice are greater in female versus male, and (2) βENaC plus ASIC2 both contribute to migration, but greater impact on female. Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males. (c). Normalized migration data in monocytes from βENaC m/m , ASIC2 −/− , and ASIC2 −/− /βENaC m/m mice are shown. Both data sets are presented as mean ± SEM and represent seven FOVs from three insets ( n = 21, except wildtype control in C where n = 21 FOVs from n = 9 inserts) and were analyzed using 2‐way ANOVA followed by Holm‐Sidak post hoc test. p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Migration, Isolation, Knock-Out, Modification, Membrane, Control

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Bone marrow macrophages from mice lacking ASIC2 plus βENaC (ASIC2 −/− /βENaC m/m , KO) are polarized towards an M1 phenotype. (a) Migration of bone marrow derived macrophages from ASIC2 −/− /βENaC m/m mice are inhibited to 55% and 65% of WT control cells from females and males, respectively. Data were analyzed using 2‐way ANOVA followed by Holm‐Sidak post hoc test. (b) Fold expression of monocyte/macrophage marker message in cultured bone marrow macrophages from males. The myeloid origin marker CD68 was decreased and M1 macrophage marker CD86 was upregulated in KO cells. The M2 marker CD206 was not significantly elevated. CD163, another marker of M2 macrophages, did not amplify in any samples. (c) Media soluble TNFα, released from M1 macrophages, was elevated in KO cell culture media. Samples were obtained from two wells from three different cell lines. Data in Panels b and d were analyzed using independent/unpaired, 2‐tailed t ‐tests. Representative images (d) and group data (e) from semiquantitative immunolabeling of CD86 and CD206 in cells show CD86, but not CD206, are elevated in KO cells. each data point represents a cluster of cells, n = 5–6 cell clusters from n = 3 images. Fluorescence is normalized to cell area. Data in panels e and f analyzed using 1‐way ANOVA followed by the Holm‐Sidak post hoc test. These findings suggest bone marrow macrophages from mice lacking βENaC and ASIC2 are polarized towards the M1 phenotype. All data are mean ± SEM. P values are provided to demonstrate confidence.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Migration, Derivative Assay, Control, Expressing, Marker, Cell Culture, Immunolabeling, Fluorescence

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Does rescue of ASIC2 or βENaC in bone marrow macrophages from ASIC2 −/− /βENaC m/m mice restore the chemotactic migration response and polarization marker expression? ASIC2 −/− /βENaC m/m (KO) male cell lines were transfected with ECFP_mouse ASIC2 or EGFP_mouse βENaC full length constructs and maintained in the presence of selection antibiotic G418 (except WT control and KO control). (a) Rescue of either ASIC2 or βENaC partially rescues the chemotactic migration response in macrophages lacking ASIC2 and βENaC. Migration data points represent 42–63 FOVs from n = 2 to 3 inserts from three trials. (b) The monocyte origin marker CD68 (b) increased with rescue of ASIC2a and M1 macrophage marker CD86 decreased (c, d), consistent with the decrease in CD68 and increase in CD86 in KO versus WT macrophages (Figure ). The C Th for GAPDH, CD68, and CD86 in KO‐EGFP control samples are within range to detect increases or decreases (20, 20, and 32, respectively). CD163 were not consistently detected in the three replicates from 2 to 5 independent trials. (d). Immunolabeling of CD86‐Viobright 515 in KO macrophages rescued with βENaC or ASIC2 are consistent with qPCR findings. Fluorescence signal of CD86 was greater than baseline EGFP/ECFP signal assessed in separate samples (not shown). (e) Soluble TNFα in the media of cultured macrophages (72 h) was decreased in βENaC, but increased in ASIC2 rescued macrophages. All data are mean ± SEM. Migration data were analyzed using 1‐way ANOVA followed by Holm‐Sidak post hoc test. Quantitative PCR were analyzed using a 1‐way ANOVA followed by Dunnett post hoc test. p values are provided to demonstrate confidence.

Article Snippet: ASIC2b , NM_007384.3 , Mm00475687_m1 , 1–2 , 105.

Techniques: Migration, Marker, Expressing, Transfection, Construct, Selection, Control, Immunolabeling, Fluorescence, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: TaqMan Gene Expression Assay Primer‐Probe Pairs for mouse ENaC Subunits.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Gene Expression, Amplification

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Primary antibodies, species, titer, and source.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques:

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: qPCR detection of α, β, γENaC, and ASIC1‐5 transcript expression in cultured bone marrow derived macrophages and freshly isolated monocytes from male and female mice. Macrophages were cultured in the presence of 20 ng/mL of CSF1. (a) PCR reactions from macrophages were separated on 3%–4% agarose gels to determine if amplicon of expected size was present (identified by arrowhead in samples with >1 product). 100 ng RNA template equivalent was used for all reactions except ASIC1, where 1000 ng was used. Three primer pair‐probe sets were tested on ASIC2 and ASIC1. The primer pair‐probe sets shown amplified a band at the expected size, in addition to 1–2 other bands. (b) Macrophage C th 's individual ENaC and ASIC transcripts from bone marrow derived macrophages 3 replicates in n = 2 trials. Thresholds at or near 35 cycles were consistently identified in all replicates for αENaC and ASIC3. Thresholds less than 39 cycles were identified for βENaC and ASIC2. γENaC amplified in only 1/6 and 4/6 replicates in male and female samples, respectively. (c) Bone marrow derived freshly isolated monocytes C th 's for individual ENaC and ASIC subunits using 75 ng RNA template equivalent. Samples from four animals were pooled. (d) Bone marrow derived freshly isolated monocyte Western blot detection of αENaC and βENaC. Samples were pooled from four animals. *Indicates statistical difference between male and female at p < 0.05 using an Uncorrected Fisher LSD Test.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Expressing, Cell Culture, Derivative Assay, Isolation, Amplification, Western Blot

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Loss of βENaC or ASIC2 inhibits bone marrow monocyte chemotactic migration. Chemotactic migration is attenuated in freshly isolated bone marrow monocytes from (a, b) βENaC hypomorph mice (βENaC m/m , 10–13 weeks of age) or (c, d) ASIC2 global knockout mice (ASIC2 −/− , 7–8 weeks of age). Monocytes were isolated in parallel from an age‐matched wildtype and modified animal within a sex then migrated overnight. Mice were used between 7 and 13 weeks of age. These findings suggest (1) migration responses in wildtype mice are greater in female versus males, and (2) βENaC and ASIC2 both contribute to migration, but differentially in the sexes. Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males. Normalized migration data are shown in Figure Representative images are shown in panels a and c and group data are shown in panels b and d. Data are mean ± SEM and represent seven FOVs from three insets ( n = 21) and were analyzed using two‐way ANOVA followed by Holm‐Sidak post hoc test. p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Migration, Isolation, Knock-Out, Modification

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Loss of ASIC2 plus βENaC on bone marrow monocyte chemotactic migration is not additive. Chemotactic migration in freshly isolated bone marrow from mice homozygous for ASIC2 global knockout and βENaC hypomorph alleles (ASIC2 −/− /βENaC m/m , 6–7 weeks). Monocytes were isolated in parallel from an age‐matched wildtype and modified animal within a sex then migrated overnight. Representative images of underside of migration membrane (a) and group data (b) in males are shown. These findings suggest (1) migration responses in wildtype mice are greater in female versus male, and (2) βENaC plus ASIC2 both contribute to migration, but greater impact on female. Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males. (c). Normalized migration data in monocytes from βENaC m/m , ASIC2 −/− , and ASIC2 −/− /βENaC m/m mice are shown. Both data sets are presented as mean ± SEM and represent seven FOVs from three insets ( n = 21, except wildtype control in C where n = 21 FOVs from n = 9 inserts) and were analyzed using 2‐way ANOVA followed by Holm‐Sidak post hoc test. p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Migration, Isolation, Knock-Out, Modification, Membrane, Control

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Bone marrow macrophages from mice lacking ASIC2 plus βENaC (ASIC2 −/− /βENaC m/m , KO) are polarized towards an M1 phenotype. (a) Migration of bone marrow derived macrophages from ASIC2 −/− /βENaC m/m mice are inhibited to 55% and 65% of WT control cells from females and males, respectively. Data were analyzed using 2‐way ANOVA followed by Holm‐Sidak post hoc test. (b) Fold expression of monocyte/macrophage marker message in cultured bone marrow macrophages from males. The myeloid origin marker CD68 was decreased and M1 macrophage marker CD86 was upregulated in KO cells. The M2 marker CD206 was not significantly elevated. CD163, another marker of M2 macrophages, did not amplify in any samples. (c) Media soluble TNFα, released from M1 macrophages, was elevated in KO cell culture media. Samples were obtained from two wells from three different cell lines. Data in Panels b and d were analyzed using independent/unpaired, 2‐tailed t ‐tests. Representative images (d) and group data (e) from semiquantitative immunolabeling of CD86 and CD206 in cells show CD86, but not CD206, are elevated in KO cells. each data point represents a cluster of cells, n = 5–6 cell clusters from n = 3 images. Fluorescence is normalized to cell area. Data in panels e and f analyzed using 1‐way ANOVA followed by the Holm‐Sidak post hoc test. These findings suggest bone marrow macrophages from mice lacking βENaC and ASIC2 are polarized towards the M1 phenotype. All data are mean ± SEM. P values are provided to demonstrate confidence.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Migration, Derivative Assay, Control, Expressing, Marker, Cell Culture, Immunolabeling, Fluorescence

Journal: Physiological Reports

Article Title: Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

doi: 10.14814/phy2.16139

Figure Lengend Snippet: Does rescue of ASIC2 or βENaC in bone marrow macrophages from ASIC2 −/− /βENaC m/m mice restore the chemotactic migration response and polarization marker expression? ASIC2 −/− /βENaC m/m (KO) male cell lines were transfected with ECFP_mouse ASIC2 or EGFP_mouse βENaC full length constructs and maintained in the presence of selection antibiotic G418 (except WT control and KO control). (a) Rescue of either ASIC2 or βENaC partially rescues the chemotactic migration response in macrophages lacking ASIC2 and βENaC. Migration data points represent 42–63 FOVs from n = 2 to 3 inserts from three trials. (b) The monocyte origin marker CD68 (b) increased with rescue of ASIC2a and M1 macrophage marker CD86 decreased (c, d), consistent with the decrease in CD68 and increase in CD86 in KO versus WT macrophages (Figure ). The C Th for GAPDH, CD68, and CD86 in KO‐EGFP control samples are within range to detect increases or decreases (20, 20, and 32, respectively). CD163 were not consistently detected in the three replicates from 2 to 5 independent trials. (d). Immunolabeling of CD86‐Viobright 515 in KO macrophages rescued with βENaC or ASIC2 are consistent with qPCR findings. Fluorescence signal of CD86 was greater than baseline EGFP/ECFP signal assessed in separate samples (not shown). (e) Soluble TNFα in the media of cultured macrophages (72 h) was decreased in βENaC, but increased in ASIC2 rescued macrophages. All data are mean ± SEM. Migration data were analyzed using 1‐way ANOVA followed by Holm‐Sidak post hoc test. Quantitative PCR were analyzed using a 1‐way ANOVA followed by Dunnett post hoc test. p values are provided to demonstrate confidence.

Article Snippet: , , , Mm00475691_m1 , 5‐6 , 79 .

Techniques: Migration, Marker, Expressing, Transfection, Construct, Selection, Control, Immunolabeling, Fluorescence, Cell Culture, Real-time Polymerase Chain Reaction