asic3  (Alomone Labs)


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    Alomone Labs asic3
    NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating <t>ASIC3.</t> a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P
    Asic3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asic3/product/Alomone Labs
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    asic3 - by Bioz Stars, 2022-11
    94/100 stars

    Images

    1) Product Images from "Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3"

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    Journal: International Journal of Oral Science

    doi: 10.1038/s41368-021-00124-6

    NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P
    Figure Legend Snippet: NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P

    Techniques Used: Transduction, Plasmid Preparation, Fluorescence, shRNA, Sequencing, DNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3
    Figure Legend Snippet: A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3

    Techniques Used:

    NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P
    Figure Legend Snippet: NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P

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

    Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P
    Figure Legend Snippet: Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P

    Techniques Used: Expressing

    2) Product Images from "Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons"

    Article Title: Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-015-0465-7

    ASICs and BK channels are co-expressed in cortical neurons. Detection of the expression of ASICs and BK channels in cortical neurons by double-staining immunofluorescence (original magnification ×400). Nuclei were counterstained with Hoechst33258 ( blue ). ASICs and BK channels were labeled with FITC ( green ) and TRITC ( red ). There is a co-expression between ASIC1, ASIC2, ASIC3, and BK channels
    Figure Legend Snippet: ASICs and BK channels are co-expressed in cortical neurons. Detection of the expression of ASICs and BK channels in cortical neurons by double-staining immunofluorescence (original magnification ×400). Nuclei were counterstained with Hoechst33258 ( blue ). ASICs and BK channels were labeled with FITC ( green ) and TRITC ( red ). There is a co-expression between ASIC1, ASIC2, ASIC3, and BK channels

    Techniques Used: Expressing, Double Immunofluorescence Staining, Labeling

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    Alomone Labs anti asic3 antibody
    Schematic diagram of the <t>ASIC3-M-CSF-M2</t> macrophage-positive feedback loop in skin fibrosis pathogenesis. Local injury leads to the formation of an acidic microenvironment, which activates fibroblast ASIC3 receptors and promotes the increase of M-CSF secretion through the PI3K/AKT signaling pathway, leading to the polarization of M2 macrophages and increasing the secretion of TGF-β1, which mediates fibroblasts to myofibroblast differentiation.
    Anti Asic3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti asic3 antibody/product/Alomone Labs
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti asic3 antibody - by Bioz Stars, 2022-11
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    Schematic diagram of the ASIC3-M-CSF-M2 macrophage-positive feedback loop in skin fibrosis pathogenesis. Local injury leads to the formation of an acidic microenvironment, which activates fibroblast ASIC3 receptors and promotes the increase of M-CSF secretion through the PI3K/AKT signaling pathway, leading to the polarization of M2 macrophages and increasing the secretion of TGF-β1, which mediates fibroblasts to myofibroblast differentiation.

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: Schematic diagram of the ASIC3-M-CSF-M2 macrophage-positive feedback loop in skin fibrosis pathogenesis. Local injury leads to the formation of an acidic microenvironment, which activates fibroblast ASIC3 receptors and promotes the increase of M-CSF secretion through the PI3K/AKT signaling pathway, leading to the polarization of M2 macrophages and increasing the secretion of TGF-β1, which mediates fibroblasts to myofibroblast differentiation.

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques:

    Activation of ASIC3 induces polarization of macrophages to M2 phenotype in vivo. A Schematic diagram of establishing rabbit ear hypertrophic scar model and experimental plan. B Immunohistochemistry analysis of ASIC3 expression in rabbit ear tissue 21 days after wounding. Scale bars, 100 μm. C Immunofluorescence staining of rabbit ear tissue 21 days after wounding showing the total (CD68+, green) and M2 subtype (CD206+, red) macrophages after treatment by PBS, DMSO, or GMQ. Scale bars, 100 μm. D , E MFI quantification of CD68 and CD206 in (C) ( n = 3). F , G Western blot and RT-qPCR analysis of rabbit ear tissue 21 days after wounding. Total CD206 protein levels and relative mRNA levels expression in control, vehicle, and GMQ groups ( n = 3). H , I Relative mRNA expressions of M1 macrophage markers (iNOS, TNF-α) as evaluated by RT-qPCR ( n = 3). Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: Activation of ASIC3 induces polarization of macrophages to M2 phenotype in vivo. A Schematic diagram of establishing rabbit ear hypertrophic scar model and experimental plan. B Immunohistochemistry analysis of ASIC3 expression in rabbit ear tissue 21 days after wounding. Scale bars, 100 μm. C Immunofluorescence staining of rabbit ear tissue 21 days after wounding showing the total (CD68+, green) and M2 subtype (CD206+, red) macrophages after treatment by PBS, DMSO, or GMQ. Scale bars, 100 μm. D , E MFI quantification of CD68 and CD206 in (C) ( n = 3). F , G Western blot and RT-qPCR analysis of rabbit ear tissue 21 days after wounding. Total CD206 protein levels and relative mRNA levels expression in control, vehicle, and GMQ groups ( n = 3). H , I Relative mRNA expressions of M1 macrophage markers (iNOS, TNF-α) as evaluated by RT-qPCR ( n = 3). Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Activation Assay, In Vivo, Immunohistochemistry, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    M2 macrophage-derived TGF-β1 induces fibroblast-to-myofibroblast differentiation. A Schematic diagram of the indirect co-culture cell model and experimental plan. B ELISA results of cytokines secreted by macrophages after ASIC3 activation by the GMQ ASIC3 activator ( n = 3). C , D Fibroblasts were treated with neutralizing antibody and evaluated for α-SMA and collagen I expression ( n = 3). E , F The contractile activities of fibroblasts treated as indicated were analyzed using fibroblast-populated collagen lattice contraction after 48 h of co-culture ( n = 3). Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: M2 macrophage-derived TGF-β1 induces fibroblast-to-myofibroblast differentiation. A Schematic diagram of the indirect co-culture cell model and experimental plan. B ELISA results of cytokines secreted by macrophages after ASIC3 activation by the GMQ ASIC3 activator ( n = 3). C , D Fibroblasts were treated with neutralizing antibody and evaluated for α-SMA and collagen I expression ( n = 3). E , F The contractile activities of fibroblasts treated as indicated were analyzed using fibroblast-populated collagen lattice contraction after 48 h of co-culture ( n = 3). Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Derivative Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing

    Activation of ASIC3 induces release of M-CSF from fibroblasts. A Experimental scheme of the cytokine antibody array. B The 80 cytokine antibody array was used to examine the expression of cytokines in cell supernatant. C Array results were analyzed by KEGG pathway enrichment analysis. D Semi-quantitative analysis of the expressions of MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 presented by heat map. E MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 expressions were determined by ELISA in cells treated as indicated for 48 h. Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: Activation of ASIC3 induces release of M-CSF from fibroblasts. A Experimental scheme of the cytokine antibody array. B The 80 cytokine antibody array was used to examine the expression of cytokines in cell supernatant. C Array results were analyzed by KEGG pathway enrichment analysis. D Semi-quantitative analysis of the expressions of MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 presented by heat map. E MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 expressions were determined by ELISA in cells treated as indicated for 48 h. Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Activation Assay, Ab Array, Expressing, Enzyme-linked Immunosorbent Assay

    ASIC3 mediates induces the release of M-CSF from fibroblasts via the PI3K/AKT/M-CSF pathway. A KEGG pathway enrichment analysis. The top 20 KEGG pathways are shown. The size of the dot reflects the gene number; the dot color indicates the q -value . B – D Western blotting and quantitative analyses of the phosphorylation of PI3K and AKT in cells treated as indicated ( n = 3). E RT-qPCR analysis of M-CSF mRNA in fibroblasts treated for 48 h with co-culture ( n = 3). F ELISA of M-CSF expression in fibroblasts treated for 48 h with co-culture ( n = 3). Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: ASIC3 mediates induces the release of M-CSF from fibroblasts via the PI3K/AKT/M-CSF pathway. A KEGG pathway enrichment analysis. The top 20 KEGG pathways are shown. The size of the dot reflects the gene number; the dot color indicates the q -value . B – D Western blotting and quantitative analyses of the phosphorylation of PI3K and AKT in cells treated as indicated ( n = 3). E RT-qPCR analysis of M-CSF mRNA in fibroblasts treated for 48 h with co-culture ( n = 3). F ELISA of M-CSF expression in fibroblasts treated for 48 h with co-culture ( n = 3). Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Western Blot, Quantitative RT-PCR, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing

    ASIC3 is highly expressed in human hypertrophic scar/keloid tissues and human primary cells. A Schematic diagram of sample source and experimental analysis. B Representative H E staining images of human normal skin, hypertrophic scar (HS), and keloid. Scale bars, 100 μm. C Immunohistochemistry of ASIC3 expression in normal skin, hypertrophic scar and keloid. Scale bars, 100 μm and 50 μm. D Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expression in human normal skin, hypertrophic scar, and keloid. Scale bars, 100 μm and 50 μm. E Western blot analysis of ASIC3 expression in normal skin, hypertrophic scar, and keloid. GAPDH served as loading control ( n = 3). F Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expressions in fibroblasts derived from normal skin, hypertrophic scar, and keloid. Scale bars, 50 μm. G MFI quantification of ASIC3 in ( F ) ( n = 3). Data are expressed as the means ± SD. ** P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: ASIC3 is highly expressed in human hypertrophic scar/keloid tissues and human primary cells. A Schematic diagram of sample source and experimental analysis. B Representative H E staining images of human normal skin, hypertrophic scar (HS), and keloid. Scale bars, 100 μm. C Immunohistochemistry of ASIC3 expression in normal skin, hypertrophic scar and keloid. Scale bars, 100 μm and 50 μm. D Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expression in human normal skin, hypertrophic scar, and keloid. Scale bars, 100 μm and 50 μm. E Western blot analysis of ASIC3 expression in normal skin, hypertrophic scar, and keloid. GAPDH served as loading control ( n = 3). F Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expressions in fibroblasts derived from normal skin, hypertrophic scar, and keloid. Scale bars, 50 μm. G MFI quantification of ASIC3 in ( F ) ( n = 3). Data are expressed as the means ± SD. ** P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Staining, Immunohistochemistry, Expressing, Immunofluorescence, Western Blot, Derivative Assay

    ASIC3 promotes scar formation in vivo. A Schematic diagram of the rabbit ear hypertrophic scar model. The model was subcutaneously administered either GMQ or DMSO. B Representative H E staining images of rabbit ear tissue at day 21 after wounding. Scale bars, 100 μm. The black boxes in the upper panel are enlarged in the lower panel. Scale bars, 50 μm. C The scar elevation index (SEI) was significantly higher in the GMQ group than in the vehicle and control groups ( n = 3). D Masson staining of rabbit ear tissue at 21 days after wounding. Scale bars, 100 μm. The black boxes in the upper panel were enlarged in the lower panel. Scale bars, 50 μm. E Immunofluorescence analysis of α-SMA (red) and collagen I (green) expressions in rabbit ear tissue 21 days after wounding ( n = 3). The dotted line indicates the epidermis. Scale bars, 100 μm. F – G Quantification of the α-SMA- and collagen I–positive areas by immunofluorescence. H – J Western blot analysis of α-SMA and collagen I expression at 7 days, 14 days, and 21 days after wounding. GAPDH served as a loading control. Data are expressed as the means ± SD ( n = 3). * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: ASIC3 promotes scar formation in vivo. A Schematic diagram of the rabbit ear hypertrophic scar model. The model was subcutaneously administered either GMQ or DMSO. B Representative H E staining images of rabbit ear tissue at day 21 after wounding. Scale bars, 100 μm. The black boxes in the upper panel are enlarged in the lower panel. Scale bars, 50 μm. C The scar elevation index (SEI) was significantly higher in the GMQ group than in the vehicle and control groups ( n = 3). D Masson staining of rabbit ear tissue at 21 days after wounding. Scale bars, 100 μm. The black boxes in the upper panel were enlarged in the lower panel. Scale bars, 50 μm. E Immunofluorescence analysis of α-SMA (red) and collagen I (green) expressions in rabbit ear tissue 21 days after wounding ( n = 3). The dotted line indicates the epidermis. Scale bars, 100 μm. F – G Quantification of the α-SMA- and collagen I–positive areas by immunofluorescence. H – J Western blot analysis of α-SMA and collagen I expression at 7 days, 14 days, and 21 days after wounding. GAPDH served as a loading control. Data are expressed as the means ± SD ( n = 3). * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: In Vivo, Staining, Immunofluorescence, Western Blot, Expressing

    Activation of ASIC3 induces polarization of M0 macrophages to M2 macrophages in vitro. A Experimental design of fibroblast and macrophage indirect co-culture assay. B Representative images of THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. C Flow cytometric analysis of CD68 in THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. D Immunofluorescence staining of CD206 (green) in experimental and control macrophages after 48 h of co-cultivation. Scale bars, 100 μm. E , F Western blot and RT-qPCR analysis of CD206 protein levels and relative mRNA levels expression in the indicated groups ( n = 3). GAPDH served as a loading control. G Expressions of CD68 and CD206 were determined by flow cytometry. H Quantification of the flow cytometry assay results (n = 3). Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: Activation of ASIC3 induces polarization of M0 macrophages to M2 macrophages in vitro. A Experimental design of fibroblast and macrophage indirect co-culture assay. B Representative images of THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. C Flow cytometric analysis of CD68 in THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. D Immunofluorescence staining of CD206 (green) in experimental and control macrophages after 48 h of co-cultivation. Scale bars, 100 μm. E , F Western blot and RT-qPCR analysis of CD206 protein levels and relative mRNA levels expression in the indicated groups ( n = 3). GAPDH served as a loading control. G Expressions of CD68 and CD206 were determined by flow cytometry. H Quantification of the flow cytometry assay results (n = 3). Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Activation Assay, In Vitro, Co-culture Assay, Derivative Assay, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry

    Activation of ASIC3 promotes fibroblast-to-myofibroblast differentiation through a process requiring macrophages. A Indirect co-culture cell model and experimental plan. B Immunofluorescence staining of α-SMA (red) and collagen I (green) in fibroblasts and control after 48 h of co-cultivation. Scale bars, 100 μm. C Western blot analysis of α-SMA in the indicated groups ( n = 3). GAPDH served as a loading control. D , E α-SMA and collagen I mRNA levels after 48 h of co-cultivation ( n = 3). F – G Wound-healing assays of fibroblast migration at 12 or 24 h after wounding ( n = 3). Scale bars are 100 μm. H , I The contractile activities of fibroblasts were analyzed using fibroblast-populated collagen lattice (FPCL); images were obtained at 0, 24, and 48 h ( n = 3). Control is untreated cells. Data are expressed as the means ± SD. * P

    Journal: Cell Death & Disease

    Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

    doi: 10.1038/s41419-022-04981-9

    Figure Lengend Snippet: Activation of ASIC3 promotes fibroblast-to-myofibroblast differentiation through a process requiring macrophages. A Indirect co-culture cell model and experimental plan. B Immunofluorescence staining of α-SMA (red) and collagen I (green) in fibroblasts and control after 48 h of co-cultivation. Scale bars, 100 μm. C Western blot analysis of α-SMA in the indicated groups ( n = 3). GAPDH served as a loading control. D , E α-SMA and collagen I mRNA levels after 48 h of co-cultivation ( n = 3). F – G Wound-healing assays of fibroblast migration at 12 or 24 h after wounding ( n = 3). Scale bars are 100 μm. H , I The contractile activities of fibroblasts were analyzed using fibroblast-populated collagen lattice (FPCL); images were obtained at 0, 24, and 48 h ( n = 3). Control is untreated cells. Data are expressed as the means ± SD. * P

    Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

    Techniques: Activation Assay, Co-Culture Assay, Immunofluorescence, Staining, Western Blot, Migration

    NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P

    Journal: International Journal of Oral Science

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    doi: 10.1038/s41368-021-00124-6

    Figure Lengend Snippet: NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P

    Article Snippet: Following fixation in cold propanol for 15 min, samples were rinsed with PBS and incubated overnight at 4 °C with primary antibodies against NGF (1:50; Abcam) or ASIC3 (1:100; Alomone), followed by incubation with secondary antibodies at 37 °C for 1 h. Fluorescence microscope (AX10 imager A2/AX10 cam HRC, Zesis) with ZEN Widefield software (Version 2012, Zesis) were used for image acquisition.

    Techniques: Transduction, Plasmid Preparation, Fluorescence, shRNA, Sequencing, DNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3

    Journal: International Journal of Oral Science

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    doi: 10.1038/s41368-021-00124-6

    Figure Lengend Snippet: A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3

    Article Snippet: Following fixation in cold propanol for 15 min, samples were rinsed with PBS and incubated overnight at 4 °C with primary antibodies against NGF (1:50; Abcam) or ASIC3 (1:100; Alomone), followed by incubation with secondary antibodies at 37 °C for 1 h. Fluorescence microscope (AX10 imager A2/AX10 cam HRC, Zesis) with ZEN Widefield software (Version 2012, Zesis) were used for image acquisition.

    Techniques:

    NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P

    Journal: International Journal of Oral Science

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    doi: 10.1038/s41368-021-00124-6

    Figure Lengend Snippet: NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P

    Article Snippet: Following fixation in cold propanol for 15 min, samples were rinsed with PBS and incubated overnight at 4 °C with primary antibodies against NGF (1:50; Abcam) or ASIC3 (1:100; Alomone), followed by incubation with secondary antibodies at 37 °C for 1 h. Fluorescence microscope (AX10 imager A2/AX10 cam HRC, Zesis) with ZEN Widefield software (Version 2012, Zesis) were used for image acquisition.

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

    Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P

    Journal: International Journal of Oral Science

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    doi: 10.1038/s41368-021-00124-6

    Figure Lengend Snippet: Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P

    Article Snippet: Following fixation in cold propanol for 15 min, samples were rinsed with PBS and incubated overnight at 4 °C with primary antibodies against NGF (1:50; Abcam) or ASIC3 (1:100; Alomone), followed by incubation with secondary antibodies at 37 °C for 1 h. Fluorescence microscope (AX10 imager A2/AX10 cam HRC, Zesis) with ZEN Widefield software (Version 2012, Zesis) were used for image acquisition.

    Techniques: Expressing

    Effect of ASA on ASIC3 expression in DRG. (A–F) Fluorescence microscope image of FB- (A, D), ASIC3- (B, E) labeled DRG neurons and their overlay (C, F) 3 weeks after MIA injection (1 week after FB injection). Arrowheads (white) indicate ASIC3 positive cells in FB-labeled neurons and yellow arrows indicate ASIC3 negative cells in FB-labeled neurons innervating the knee joint. Scale bar: 100 μm. (G) Effect of ASA on ASIC3 expression in DRG. ASIC3 expression was counted in FB-labeled neurons in L3-4 DRGs and quantified as the percentage of total FB-labeled neurons. Means ± S.E.M, N = 4–6, ∗: p

    Journal: Heliyon

    Article Title: Mechanism of aspirin-induced inhibition on the secondary hyperalgesia in osteoarthritis model rats

    doi: 10.1016/j.heliyon.2020.e03963

    Figure Lengend Snippet: Effect of ASA on ASIC3 expression in DRG. (A–F) Fluorescence microscope image of FB- (A, D), ASIC3- (B, E) labeled DRG neurons and their overlay (C, F) 3 weeks after MIA injection (1 week after FB injection). Arrowheads (white) indicate ASIC3 positive cells in FB-labeled neurons and yellow arrows indicate ASIC3 negative cells in FB-labeled neurons innervating the knee joint. Scale bar: 100 μm. (G) Effect of ASA on ASIC3 expression in DRG. ASIC3 expression was counted in FB-labeled neurons in L3-4 DRGs and quantified as the percentage of total FB-labeled neurons. Means ± S.E.M, N = 4–6, ∗: p

    Article Snippet: After washing with 0.1 M PBS (pH 7.4), sections were incubated at 4 °C overnight with rabbit anti-ASIC3 antibody (1:500, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Fluorescence, Microscopy, Labeling, Injection