Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: High-fat diet attenuates ventilator-induced lung injury (VILI) in mice. A : H&E staining (magnification, ×200 and ×400) and lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin. The relative values are normalized to GAPDH; n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. HFD, hight-fat diet; MV, mechanical ventilation; NCD, normal chow diet; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Uptake of serum exosome (S-Exo), adipose tissue exosome (AT-Exo), and adipose-derived stem cell exosome (ADSC-Exo) by lung tissue and pulmonary microvascular endothelial cells (PMVECs) and the optimization of the optimal exosome intervention concentrations in vivo and in vitro. A : uptake of S-Exo, AT-Exo, and ADSC-Exo by lung tissue. B : uptake of S-Exo, AT-Exo, and ADSC-Exo by PMVECs. In the fluorescence microscopy pictures, FITC-labeled CD31 was used to label the vascular endothelial cells (green), PKH26 was used to label the exosomes (red), and DAPI was used to detect the nucleus (blue). C and E : effects of three exosomes (S-Exo, AT-Exo, and ADSC-Exo) at different concentrations (0, 25, 50, and 100 μg/mL) on the expression of transient receptor potential vanilloid 4 (TRPV4) under mechanical ventilation in mice. The mRNA level of TRPV4 was evaluated by quantitative real-time PCR (qRT-PCR) and the protein level of TRPV4 was evaluated by Western blotting (WB). D and F : effects of three exosomes (S-Exo, AT-Exo, and ADSC-Exo) at different concentrations (0, 25, 50, and 100 μg/mL) on the expression of TRPV4 under 18% cyclic stretching in PMVECs. The mRNA level of TRPV4 was evaluated by qRT-PCR and the protein level of TRPV4 was evaluated by WB. n = 4 samples from each group assayed in triplicate. All results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; CS, cyclic stretching.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, In Vitro, Fluorescence, Microscopy, Labeling, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Exploring the role of serum exosome (S-Exo) in ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400), lung injury scores are used for the semiquantitative analysis of lung histopathologic damage. B : The wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: The effect of adipose tissue exosome (AT-Exo) on ventilator-induced lung injury (VILI) in vivo. A : hematoxylin and eosin (HE) staining (magnification, ×200 and ×400); lung injury scores are used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH; n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. CM, conditioned media; MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: The effect of adipose-derived stem cell exosome (ADSC-Exo)-based treatment on ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400); lung injury scores are used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing; ADSC-CM, ADSC conditioned media.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Serum exosome (S-Exo) partly suppresses pulmonary microvascular endothelial cell (PMVEC) inflammation and promotes barrier function after 18% cyclic stretching (CS) through inhibiting transient receptor potential vanilloid 4 (TRPV4)/Ca 2+ signaling in vitro. A : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. C : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. D and E : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vitro, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Adipose tissue exosome (AT-Exo) and adipose-derived stem cell exosome (ADSC-Exo) promote pulmonary microvascular endothelial cell (PMVEC) barrier function and suppress inflammation after 18% cyclic stretching (CS) through inhibiting transient receptor potential vanilloid 4 (TRPV4)/Ca 2+ signaling in vitro. A and E : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B and F : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. C and G : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. D and H : intracellular calcium ions are detected by flow cytometry (FCM); n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. ADSC-CM, ADSC conditioned media.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vitro, Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Effects of the combination of adipose-derived stem cell exosome (ADSC-Exo) and transient receptor potential vanilloid 4 (TRPV4) agonist (GSK1016790A)/antagonist (HC-067047) on ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400); lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Effects of the combination of adipose-derived stem cell exosome (ADSC-Exo) and transient receptor potential vanilloid 4 (TRPV4) agonist (GSK1016790A)/antagonist (HC-067047) on 18% cyclic stretching (CS) in vitro. A : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. C : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. D : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vitro, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: One-shot treatment with adipose-derived stem cell exosome (ADSC-Exo) alleviates pulmonary endothelial barrier hyperpermeability and inflammation response after ventilator-induced lung injury (VILI) in vivo and in vitro. A : H&E staining (magnification, ×200 and ×400); lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F and J : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G and K : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. H : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. I : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 mice or cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; CS, cyclic stretching.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, In Vitro, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: A . Heatmaps of miRs show differences in expression levels between WT and TRPV4 KO skin tissues implanted with 50 kPa PA hydrogels (n = 5 mice/group; pooled samples). B . A scatter plot displays the DEGs based on their log2 fold change values. C . Bar graph shows the fold-change in miR-146a expression between WT and TRPV4 KO skin tissues. D-E . Spinning disc confocal images reveal calcium influx (red) in WT and TRPV4 KO macrophages under untreated conditions (UT), with LPS (100 ng/ml) alone, and with LPS plus TRPV4-specific agonist, GSK101 (100 nM), treatment ( D ). The quantification of the fluorescence intensity of the cells (n = 50 cells/condition) under all conditions is shown in E . F-G . Spinning disc confocal images display calcium influx (red) in WT and TRPV4 KO macrophages under LPS treatment, and after LPS treatment for 24 h followed by GSK101 perfusion for 10 mins ( F ). The quantification of the fluorescence intensity of the cells (n = 50 cells/condition) from F is represented in G . H-O . WT and TRPV4 KO macrophages were treated with 0, 100, 250, and 500 ng/ml of LPS for a dose kinetics study and with 100 ng/ml of LPS for 0, 6, 12, and 24 h for a time kinetics study. The quantification of RT-qPCR analysis shows the expression of miR-146a under different treatment conditions. Plots from RT-qPCR analysis of mature miR-146a expression in macrophages ( H-K ) and pre-miR-146a ( L-O ) under varying doses and time points are presented. Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: WT and TRPV4 KO macrophages were either left untreated or treated with LPS (100 ng/ml) alone, or with LPS in combination with 1, 5, or 25 μM of the TRPV4-specific antagonist GSK219. A-B . The quantification of RT-qPCR analysis reveals the expression levels of mature miR-146a in thio-MPMs in A and in BMDMs in B . The expression levels of pre-miR-146a in BMDMs are shown in C . D . Bar graph displays the fold-change of TRAF6 mRNA in WT and TRPV4 KO macrophages treated with or without 100 ng/ml of LPS for 24 hours. WT and TRPV4 KO macrophages were seeded on 1 and 50 kPa polyacrylamide hydrogel plates coated with collagen (10 μg/ml) and then treated with or without 100 ng/ml of LPS for 24 hours. E-F . Bar graphs show the expression levels of mature miR-146a ( E ) and pre-miR-146a ( F ) in thio-MPMs seeded on plates of varying stiffness. G-H . The plots display the expression levels of mature miR-146a ( G ) and pre-miR-146a ( H ) in BMDMs seeded on plates of varying stiffness. Three experimental repeats were conducted for each experiment, and data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: Quantitative RT-PCR, Expressing, Comparison

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: The schematic diagram illustrates adenovirus (Ad) expression constructs encoding WT-TRPV4 and three mutant forms with deletions of N-terminal residues 1 to 130, 1 to 30, or 100 to 130. PRD represents the proline-rich domain; ANK denotes ankyrin repeats, alongside the pore domain and the N and C terminals of the TRPV4 protein. B . Quantification of RT-qPCR analysis reveals the miR-146a fold-change in TRPV4 KO macrophages transfected with Ad-Vec, Ad-WT-TRPV4, and the three mutant constructs (Ad-TRPV4 del 1-30, Ad-TRPV4 del 1-130, and Ad-TRPV4 del 100-130) under either untreated conditions or after LPS (100 ng/ml) treatment for 24 h. Experiments were repeated three times, and data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test; **p < 0.01, ***p < 0.001.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: Expressing, Construct, Mutagenesis, Quantitative RT-PCR, Transfection, Comparison

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: A . WT and TRPV4 KO macrophages were treated with LPS (100 ng/ml) plus IFNy (20 ng/ml) for 0.5, 1, 2, 6, and 24 h or kept untreated for the control group. Immunoblotting and quantification of phosphorylated P65, Stat1, P38, JNK1/2, ERK and AKT relative to their total protein levels are shown. Actin was used as loading control. All experiments performed three times and in two biological replicates. B-G . Bar graphs show quantification of results from A: p-P65 ( B ), p-P38 ( C ), p-Stat1 ( D ), p-JNK ( E ), p-ERK ( F ), and p-AKT ( G ). Data analyzed using One-way ANOVA followed by Bonferroni’s multiple comparison test; * p < 0.05, *** p < 0.001; ns, not significant.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: Schematic representation of the transcription start sites of the miR-146a promoter, encompassing the 10 CpG islands (denoted by blue crosses) that were analyzed in our study. Four amplicons were designed to cover the CpG sites of interest, designated by blue arrows. B . Percentage of methylation of the 10 CpG sites for conditions WT-UT, WT-100 ng/ml LPS treated (24 h), TRPV4 KO-UT, and TRPV4 KO-100 ng/ml LPS treated (24 h) depicted by a line graph. C . Quantification of the percentage of methylation for each condition in the miR-146a gene depicted by a bar plot. D . Immunoblotting of acetylated histone H3 relative to total histone levels from WT and TRPV4 KO macrophages treated with or without LPS (100 ng/ml) for 24 and 48 h. E . Quantification of the data from D plotted as bar plots, n = 3 independent experiments. F . Immunoblot shows acetylated histone H3 in WT and TRPV4 KO macrophages treated with LPS (100 ng/ml) in the presence or absence of the HDAC inhibitor TSA. The control group contained cells that remained untreated (UT). G . Quantification of the data from the experiment presented in F shown by a bar plot, n = 3 independent experiments. H . Quantification of results from RT-qPCR analysis shows the expression of miR-146a from WT and TRPV4 KO macrophages treated with LPS in the presence or absence of the HDAC inhibitor TSA and in the control untreated (UT) group. Data analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test; *p < 0.05, **p < 0.01; ns, not significant.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: Methylation, Western Blot, Quantitative RT-PCR, Expressing, Comparison

Journal: bioRxiv

Article Title: Mechanisms underlying TRPV4-mediated regulation of miR-146a expression

doi: 10.1101/2024.04.03.587984

Figure Lengend Snippet: Combined fluorescent in situ hybridization (FISH) and immunofluorescence (IF) were carried out on 7 μm aortic root sections from ApoE KO and ApoE:TRPV4 DKO mice fed either chow or a high-fat diet. A . Representative IF-FISH images of the tissues hybridized with a miR-146a probe (red), co-stained with a primary antibody for CD68 and an Alexa Fluor conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Tissues stained in the absence of the miR-146a probe and Alexa Fluor conjugate secondary antibody were used as negative controls. N = 5 mice per group, with 6 images taken for each group. B . Quantification of the CD68-positive macrophages in the aortic root tissues under different conditions from figure A. C . miR-146a expression quantified for different conditions from figure A. D . Quantification of co-stained areas in tissues under different conditions. One-way ANOVA followed by Bonferroni’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: We used the following adenovirus constructs generated by Vector Biolabs: Ad (RGD)-mouseTRPV4-WT (Ad-TRPV4-WT), three deletion constructs of different lengths of mouse WT TRPV4 gene (Ad-TRPV4-Δ1-30, Ad-TRPV4-Δ1-130, and Ad-TRPV4-Δ100-130), and Ad-Vec control.

Techniques: In Situ Hybridization, Immunofluorescence, Staining, Expressing, Comparison

Journal: Materials Today Bio

Article Title: High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

doi: 10.1016/j.mtbio.2024.101058

Figure Lengend Snippet: Higher extracellular viscosity enhanced the translocation of TRPV4 to the cell membrane, thereby promoting calcium influx and osteogenic lineage in hMSCs. (A) The time course of intracellular Ca 2+ levels, quantified in terms of Fluo 8-am, in hMSCs with and without a 3-h pre-treatment of the 10 μM TRPV4 inhibitor (GSK205), followed by stimulation with a high-viscosity medium (68.14 cP). Cell culture in DMEM (0.98 cP) serves as control. (B) Time-dependent relative changes in hMSCs' area, corresponding to A. Data represent mean ± SD (n = 10). (C – E) The surface labeling of TRPV4 (in green) on hMSCs, with and without prior treatment with 10 μM of GSK205 (a TRPV4 antagonist), followed by stimulation with a highly viscous medium (68.14 cP) for 1 h. The surface labeling procedure included the blocking of non-permeabilized fixed hMSCs with BSA for 1 h, followed by a 1-h incubation with anti-TRPV4 and two PBS washes. Finally, the cells were incubated with an Alexa Fluor 488-conjugated antibody for 1 h. Scale bars = 50 μm. (F) Quantification of the number and the intensity of TRPV4 fluorescence spots on the cell membrane, as depicted in (C–E). Data represent mean ± SD (n = 15). (G – I) Representative micrographs of hMSCs, cultured in differentiation medium with and without treatment with 10 μM of GSK205 followed by stimulation with a highly viscous medium (68.14 cP) for 14 days. The cells were stained to visualize the presence of ALP activity (in purple) and lipids (Oil Red O, red). Scale bars = 50 μm. (J) The percentage of osteogenesis (ALP-positive cells) and adipogenesis (Oil Red O-positive cells) in hMSCs, as depicted in (I). Data represent mean ± SD (n = 3; i.e., three replicated independent experiments). * for p < 0.05 and ** for p < 0.01. ; N.S.: Not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used in this experiment were 1:100 dilution of anti-TRPV4 rabbit antibody (65893, Cell Signaling), 1:50 dilution of anti-NFATc1 mouse monoclonal antibody (sc-7294, Santa Cruz Biotechnology), 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex), and 1:200 dilution of anti-YAP rabbit polyclonal antibody (GTX129151, GeneTex).

Techniques: Viscosity, Translocation Assay, Membrane, Cell Culture, Control, Labeling, Blocking Assay, Incubation, Fluorescence, Staining, Activity Assay

Journal: Materials Today Bio

Article Title: High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

doi: 10.1016/j.mtbio.2024.101058

Figure Lengend Snippet: Higher extracellular viscosity promoted membrane translocation of TRPV4 and the nuclear translocation of NFATc1. (A) Immunofluorescence micrographs displaying NFATc (in red), TRPV4 on membrane (in green), and the nucleus (in blue), in hMSCs with and without prior treatment with GSK205 (10 μM), and subsequently stimulated with a highly viscous medium (68.14 cP) for 1 h. Scale bars = 50 μm. (B) Cell area, (C) mean fluorescence intensity of TRPV4, and (D) the ratio of NFATc in the nucleus to that in the cytoplasm of hMSC, as indicated in (A). Data represent mean ± SD (n = 15). * for p < 0.05 and ** for p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used in this experiment were 1:100 dilution of anti-TRPV4 rabbit antibody (65893, Cell Signaling), 1:50 dilution of anti-NFATc1 mouse monoclonal antibody (sc-7294, Santa Cruz Biotechnology), 1:200 dilution of anti-paxillin rabbit polyclonal antibody (GTX125891, GeneTex), and 1:200 dilution of anti-YAP rabbit polyclonal antibody (GTX129151, GeneTex).

Techniques: Viscosity, Membrane, Translocation Assay, Immunofluorescence, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: High-fat diet attenuates ventilator-induced lung injury (VILI) in mice. A : H&E staining (magnification, ×200 and ×400) and lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin. The relative values are normalized to GAPDH; n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. HFD, hight-fat diet; MV, mechanical ventilation; NCD, normal chow diet; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Uptake of serum exosome (S-Exo), adipose tissue exosome (AT-Exo), and adipose-derived stem cell exosome (ADSC-Exo) by lung tissue and pulmonary microvascular endothelial cells (PMVECs) and the optimization of the optimal exosome intervention concentrations in vivo and in vitro. A : uptake of S-Exo, AT-Exo, and ADSC-Exo by lung tissue. B : uptake of S-Exo, AT-Exo, and ADSC-Exo by PMVECs. In the fluorescence microscopy pictures, FITC-labeled CD31 was used to label the vascular endothelial cells (green), PKH26 was used to label the exosomes (red), and DAPI was used to detect the nucleus (blue). C and E : effects of three exosomes (S-Exo, AT-Exo, and ADSC-Exo) at different concentrations (0, 25, 50, and 100 μg/mL) on the expression of transient receptor potential vanilloid 4 (TRPV4) under mechanical ventilation in mice. The mRNA level of TRPV4 was evaluated by quantitative real-time PCR (qRT-PCR) and the protein level of TRPV4 was evaluated by Western blotting (WB). D and F : effects of three exosomes (S-Exo, AT-Exo, and ADSC-Exo) at different concentrations (0, 25, 50, and 100 μg/mL) on the expression of TRPV4 under 18% cyclic stretching in PMVECs. The mRNA level of TRPV4 was evaluated by qRT-PCR and the protein level of TRPV4 was evaluated by WB. n = 4 samples from each group assayed in triplicate. All results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; CS, cyclic stretching.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, In Vitro, Fluorescence, Microscopy, Labeling, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Exploring the role of serum exosome (S-Exo) in ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400), lung injury scores are used for the semiquantitative analysis of lung histopathologic damage. B : The wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: The effect of adipose tissue exosome (AT-Exo) on ventilator-induced lung injury (VILI) in vivo. A : hematoxylin and eosin (HE) staining (magnification, ×200 and ×400); lung injury scores are used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH; n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. CM, conditioned media; MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: The effect of adipose-derived stem cell exosome (ADSC-Exo)-based treatment on ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400); lung injury scores are used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing; ADSC-CM, ADSC conditioned media.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Serum exosome (S-Exo) partly suppresses pulmonary microvascular endothelial cell (PMVEC) inflammation and promotes barrier function after 18% cyclic stretching (CS) through inhibiting transient receptor potential vanilloid 4 (TRPV4)/Ca 2+ signaling in vitro. A : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. C : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. D and E : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: In Vitro, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Adipose tissue exosome (AT-Exo) and adipose-derived stem cell exosome (ADSC-Exo) promote pulmonary microvascular endothelial cell (PMVEC) barrier function and suppress inflammation after 18% cyclic stretching (CS) through inhibiting transient receptor potential vanilloid 4 (TRPV4)/Ca 2+ signaling in vitro. A and E : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B and F : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. C and G : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. D and H : intracellular calcium ions are detected by flow cytometry (FCM); n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. ADSC-CM, ADSC conditioned media.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vitro, Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Effects of the combination of adipose-derived stem cell exosome (ADSC-Exo) and transient receptor potential vanilloid 4 (TRPV4) agonist (GSK1016790A)/antagonist (HC-067047) on ventilator-induced lung injury (VILI) in vivo. A : H&E staining (magnification, ×200 and ×400); lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. n = 4 mice from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; SB, spontaneously breathing.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: Effects of the combination of adipose-derived stem cell exosome (ADSC-Exo) and transient receptor potential vanilloid 4 (TRPV4) agonist (GSK1016790A)/antagonist (HC-067047) on 18% cyclic stretching (CS) in vitro. A : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. B : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. C : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. D : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vitro, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Adipose-derived exosomes protect the pulmonary endothelial barrier in ventilator-induced lung injury by inhibiting the TRPV4/Ca 2+ signaling pathway

doi: 10.1152/ajplung.00255.2019

Figure Lengend Snippet: One-shot treatment with adipose-derived stem cell exosome (ADSC-Exo) alleviates pulmonary endothelial barrier hyperpermeability and inflammation response after ventilator-induced lung injury (VILI) in vivo and in vitro. A : H&E staining (magnification, ×200 and ×400); lung injury scores were used for the semiquantitative analysis of lung histopathological damage. B : the wet/dry (W/D) weight ratio. C : protein concentrations in bronchoalveolar lavage fluid (BALF). D : myeloperoxidase (MPO) activity in lung tissue. E : proinflammatory cytokines TNF-α and IL-6 in BALF. F and J : quantitative real-time PCR (qRT-PCR) analysis shows the gene expression of TNF-α, IL-6, transient receptor potential vanilloid 4 (TRPV4), β-catenin, and VE-cadherin normalized to the gene expression of GAPDH. G and K : Western blot (WB) analysis shows the protein expression of TNF-α, IL-6, TRPV4, β-catenin, and VE-cadherin, and the relative values are normalized to GAPDH. H : proinflammatory cytokines TNF-α and IL-6 in cell culture supernatant. I : intracellular calcium ions are detected by flow cytometry (FCM). n = 4 mice or cultures from each group assayed in triplicate. The results are expressed as means ± SE. * P < 0.05. MV, mechanical ventilation; CS, cyclic stretching.

Article Snippet: Furthermore, mice were treated with the combination of ADSC-Exo and TRPV4 agonist GSK1016790A (6 nM; MedChemExpress) or TRPV4 antagonist HC-067047 (20 nM; MedChemExpress) at the onset of MV ( ) to confirm the involvement of TRPV4 signaling in adipose-derived exosomes-mediated protection against VILI in vivo.

Techniques: Derivative Assay, In Vivo, In Vitro, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: Primer sequences for the genes of interest used in the current study

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Sequencing

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: TRPV4 expression increased during the inflammatory/acute phase of SCI. A, Schematic showing SCI instrument and method. B, Schematic illustration showing TPM imaging after SCI. Representative immunohistochemistry images performed on longitudinal sections at epicenter of the damage for TRPV4 (C), and NF and RECA-1 (D) at 3 h, 8 h, and 1, 3, 5, and 7 d after moderate static compression (35 g/5 min) SCI (Carl Zeiss microscope, 3 fields/slide, n = 3/time point). SCI rat sectional slice labeled with 10 μm SCa1-IREF for 40 min. In situ Ca2+ levels were determined in the transverse spinal cord using TPM at similar time points after SCI at the epicenter of the damage. E, Two-photon excited fluorescence was collected using 750 nm excitation and emission windows at 400–430 nm (Ch1) and 500–600 nm (Ch2). Total RNA was prepared from the epicenter of the damage collected 3 h, 8 h, and 1, 3, 5, 7, 14, 21, and 28 d after SCI to determine the expression of TRPV4 (F), IL-6 (H), and Pacsin-3 (I) (n = 4–6/time point, performed in triplicates). G, Quantification of in situ Ca2+ levels. GAPDH was used as internal controls for qRT-PCR. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. $p < 0.05, $$$p < 0.001 compared with HPI-3. #p < 0.05 compared with HPI-8. @p < 0.05, @@p < 0.01, @@@p < 0.001 compared with DPI-1. ∧p < 0.05, ∧∧p < 0.01 compared with DPI-3. np < 0.05, nnp < 0.01 compared with DPI-5. gp < 0.05, gggp < 0.01 compared with DPI-7. xp < 0.05, xxxp < 0.01 compared with DPI-21 by one-way ANOVA Tukey's post test.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Expressing, Imaging, Immunohistochemistry, Microscopy, Labeling, In Situ, Fluorescence, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: TRPV4 KO mice display reduced hyperalgesia, improved functional recovery, enhanced neuroprotection, and endothelial preservation after SCI. A, Representative images of H&E staining at DPI-28 after SCI. B, Functional recovery was assessed in open-field testing by using the 9-point BMS locomotor test at 1, 3, 7, 14, 21, and 28 d after SCI (n = 10/group). C, Nociception was evaluated using hotplate test. D, SCI-induced hypersensitivity (decrease in reaction latency time) was assessed at pre-SCI (basal), 1, 3, 7, 14, 21, and 28 d after SCI (n = 6/group). Samples from WT (injury) or TRPV4 KO (injury) were prepared at DPI-28 as described in Materials and Methods. Representative sections of angiopoietin-1 (ANG-1, white), NT-3 (red), and BDNF (green) in WT (E) and KO (F). Representative section of vWF (G; green), neural/glial antigen NG-2 (H; green), α-SMA (H; red), Connexin-43 (I; green), Tuj-1 (I; magenta); growth cone (green; J), and NeuN (magenta, J). Bar charts represent the IMV (fluorescence) for corresponding protein as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Functional Assay, Preserving, Staining, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: Primary antibody information used for immunoblot analysis

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: Primary antibody information used for immunohistochemistry analysis

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Immunohistochemistry

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: TRPV4 expression is linked with injury severity and inflammation. Schematic showing method for injury-dependent expression and impounder of different weight (A,B). H&E staining was performed on the longitudinal section after mild (20 g/5 min), moderate (35 g/5 min), and severe (50 g/5 min) injury (C). Total RNA was prepared from spinal cord tissues at the rostral, epicenter, and caudal of the damage collected at 1 d after SCI (mild, moderate, and severe compression) to determine the expression of TRPV4 (D), IL-6 (E), Pacsin3 (F), and occludin (G). RNA expression was determined at the epicenter at 1 d after SCI (mild, moderate, and severe) (n = 2 or 3/group performed in triplicates). TRPV4 (I) and NF and RECA-1 (L) IHC was performed after mild (20 g), moderate (35 g), and severe (50 g) injury (Carl Zeiss microscope, 3 fields/slide, n = 3/time point). In situ Ca2+ levels were determined using TPM at 8 h after SCI (mild, moderate, and severe) at the epicenter of the damage (J). Quantification of in situ Ca2+ levels (H). Colocalization of TRPV4 with CD-31, an endothelial marker, and Iba-1, a microglial marker (K). Total RNA was prepared from spinal cord tissues after spinal cord hemisection to determine the expression of TRPV4 (M), IL-6 (N), and Pacisn3 (O) (n = 4 or 5/group). GAPDH was used as internal controls for qRT-PCR. Data are mean ± SEM performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with 20 g injury group. #p < 0.05, ##p < 0.01, ###p < 0.001 compared with 35 g injury group by one-way ANOVA Tukey's post test.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Expressing, Staining, RNA Expression, Microscopy, In Situ, Marker, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: TRPV4 activation causes endothelial remodeling/damage, whereas TRPV4 inhibition protects SCI-induced endothelial damage. TRPV4 and CD-31 (A) immunocytochemistry was performed on fixed hCMEC/D3 BBB cell line as described in Materials and Methods. Quantification of TRPV4 and CD-31 fluorescence (B,C). TRPV4 and vWF immunocytochemistry was performed on fixed HUVECs (D) and its quantification (E,F). GSK1016790A (50 pmol, 10 μl) was injected into the spinal cord of the rats as mentioned in Materials and Methods. Samples from sham, GSK1016790A, vehicle (injury), or GSK1016790A (injury) were prepared 28 d after injection/injury. Representative images of laminin (magenta) and RECA-1 (G; green), GFAP (H; green), Iba-1 (H; red), and NFs (I; red). J–L, Bar charts represent the fluorescence IMV for corresponding protein as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). Samples from sham or injured untreated (injury) or after RN-1734 (5 mg/kg) treatment were prepared 1 d after moderate injury (35 g/5 min). Representative transverse section of TRPV4 (M) and ZO-1 (N) (3 fields/slide, n = 3) and fluorescence IMV for corresponding protein (P,Q). Total RNA from sham, vehicle (injury), or RN-1734-treated (5 mg/kg, i.p.) samples was prepared DPI-1 after injury. RT-PCR results are showing relative expression levels of TRPV4 (S), Pacsin3 (T), inducible nitric oxide synthase (U), IL-6 (V), and occludin (W). In situ Ca2+ levels were determined in the transverse spinal cord using TPM at 8 h after SCI at the epicenter of the damage (O). Quantification of in situ Ca2+ levels (R). Data are mean ± SEM (n = 2 or 3/group performed in triplicates). *p < 0.05, **p < 0.01, compared with sham group. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with GSK group. #p < 0.05 (B,C,E). #p < 0.05, ##p < 0.01, ###p < 0.001 compared with sham group. *p < 0.05, **p < 0.01, compared with injury group (P–W). *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with GSK group (J–L) by one-way ANOVA Tukey's post test.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Activation Assay, Inhibition, Immunocytochemistry, Fluorescence, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: Reduced endothelial damage and inflammation after SCI in TRPV4 KO mice. Representative images of H&E staining at DPI-1 after SCI (A). Representative whole spinal cords show Evans blue dye extravasation of the spinal cord 1 d after SCI (B) and its quantification (C). Total RNA from sham (black bar), wild (red bar), or TRPV4 KO (blue bar) samples was prepared DPI-1 after the injury as described in Materials and Methods. RT-PCR results show relative expression levels of occludin (D), VEGF (E), IL-1β (F), IL-6 (G), TNF-α (H), macrophage-1 antigen (Mac-1) (I), chemokine (C-C motif) ligand 2 (CCL-2) (J), and chemokine (C-C motif) ligand 3 (CCL-3) (K) after injury (n = 2 or 3/group performed in triplicates). GAPDH was used as internal controls for qRT-PCR. TUNEL assay was performed at DPI-1 (L). Quantification of TUNEL-positive cells (M). SCI-induced mouse sectional slice labeled with 10 μm SCa1-IREF for 40 min. In situ Ca2+ levels were determined in the longitudinal spinal cord using TPM at 8 h after SCI at the epicenter of the damage. Two-photon excited fluorescence was collected using 750 nm excitation and emission windows at 400–430 nm (Ch1) and 500–600 nm and its quantification (Ch2) (N,O). Representative images for ZO-1 (magenta) and occludin (Q; green) and fluorescence IMV for corresponding protein (P). ###p < 0.001 compared with sham group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with WT-injury group (D–K,O) by one-way ANOVA Tukey's post test. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group (C,M,P) by unpaired t test.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, TUNEL Assay, Labeling, In Situ, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

doi: 10.1523/JNEUROSCI.2035-19.2020

Figure Lengend Snippet: TRPV4 KO mice exhibit less fibrotic and glial scarring after SCI. Samples from WT (injury) or TRPV4 KO (injury) were prepared at DPI-28 as described in Materials and Methods. Representative merges images (longitudinal) for Collagen IV (A; white), laminin (B; magenta), GFAP (C; green), NF (C; magenta), Iba-1 (D; magenta), CD-206 (E; green), CD-68 (F, green), and TGF-β1 (F; magenta) at DPI-28. Bar charts represent the fluorescence IMV for Collagen IV (G), Iba-1 (H), laminin (I), CD-206 (J), GFAP and NF (K), and TGF-β1 and CD-68 (L) as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). M, Western blots of NFs, Iba-1, Connexin-43, GFAP, and CD-31 expression at 28 d after injury. Quantification of immunoblot for NF (N), Iba-1 (O), Connexin-43 (P), GFAP (Q), and CD-31 (R) was performed using ImageJ. Actin was used as internal controls for Western blot (n = 2 or 3/group). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group by unpaired t test.

Article Snippet: Mice heterozygous (B6.129X1-Trpv4) for Trpv4 deficiency ( Suzuki et al., 2003 ) were purchased from RIKEN BioResource Centre and intercrossed to generate Trpv4 KO mice.

Techniques: Fluorescence, Western Blot, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitation (IP) analysis of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in mCCDcl1 cells and mouse kidney. A: immunoblots of mCCDcl1 cell homogenates show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). B: immunoblots of mouse kidney homogenates also show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously ((20), Fig. 1). CAV-1 (22 kD) protein band is demonstrated in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitaton (IP) analysis of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney. Immunoblots of mCCDcl1 cell homogenates show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (A). Immunoblots of mouse kidney homogenates also show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (B). Endogenous immunoprecipitaton of IK1 (C) or BKα (D) followed by blotting for CAV-1, TRPV4, and each of the KCa channels, in mCCDcl1 cells, showed enrichment of CAV-1, TRPV4, BKα, SK3, and BKα or IK1. Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously [(20) Fig. 1]. CAV-1 (22 kD) was verified in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Effect of caveolin-1 (CAV-1) knockdown on transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated (GSK101) intracellular Ca2+ ([Ca2+]i) response to blockers of TRPV4 and KCa channels. The normal peak [Ca2+]i response to TRPV4 activation (GSK101) is markedly reduced from 597 ± 45 nM (n = 70) to 231 ± 47 nM (n = 85) following CAV-1 siRNA treatment (A and B). Furthermore, the apparent initial rate of TRPV4-mediated Ca2+ influx was markedly depressed from 108 ± 27 nM/min (n = 70) in normal conditions to 42 ± 16 nM/min (n = 85) in the presence of CAV-1 siRNA (C), reflecting a markedly depressed level of TRPV4 activation. The subsequent panels demonstrate that the typical normal peak elevation in [Ca2+]i following TRPV4 activation is markedly reduced upon inhibition of each KCa channel following CAV-1 knockdown. Iberiotoxin (IbTX) treatment reduces the peak [Ca2+]i for BK inhibition from 227 ± 34 nM (n = 73) to 84 ± 33 nM (n = 59) after CAV-1 siRNA treatment (D and E). TRAM-34 treatment reduces the peak [Ca2+]i for IK1 inhibition from 312 ± 31 nM (n = 90) to 81 ± 12 nM (n = 95) after CAV-1 siRNA treatment (F and G). Apamin treatment reduces the peak [Ca2+]i for SK3 inhibition from 414 ± 60 nM (n = 72) to 196 ± 23 nM (n = 60) after CAV-1 siRNA treatment (H and I). Therefore, after CAV-1 siRNA treatment the response for each of the KCa channels to TRPV4 activation is markedly depressed. ***P < 0.001. BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Activation Assay, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Representative immunofluorescence images show colocalization of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in collecting duct mCCDcl1 cells in mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for TRPV4 (TRPV4, green) and SK3 (SK3-ATTO-594, red) show strong colocalization as apparent in the merged image (Merged, yellow) (A). Similarly, immunostaining for TRPV4 (red) and BKα (green) (B) and for TRPV4 (red) and IK1 (green) (C) demonstrated noted colocalization in the merged images (Merged, yellow). Mouse kidney CCD immunostained for TRPV4 (TRPV4, green) and SK3 (SK3, ATTO-594, red) display noted colocalization in the merged images (Merged, yellow) (D). In a similar manner, immunostaining for TRPV4 (TRPV4, red) and BKα (BKα, green) (E) and for TRPV4 (TRPV4, red) and IK1 (IK1, green) (F) likewise demonstrated apparent colocalization in the merged images (Merged, yellow). Areas of more diffuse staining are also apparent in some cases, especially for IK1 (see F). Note that we verified the primary antibodies using blocking peptides in an earlier publication [(20) Figs. 1 and ​and4].4]. All the immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining, Staining, Blocking Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Immunofluorescence images demonstrates colocalization of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) showed strong colocalization in the merged image (Merged, yellow) (A). Similarly, immunostaining for CAV-1 with each of the KCa channel demonstrated robust colocalization in the merged images as shown for CAV-1 (green) and SK3 (red) (B), CAV-1 (red) and BKα (green) (C), and CAV-1 (red) and IK1 (green) (D). Diffuse localization was also apparent, especially for CAV-1 and IK1 (D). Mouse kidney CCD immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) also displayed robust colocalization as shown in the merged image (Merged, yellow) (E). Similarly, immunostaining for CAV-1 and each of the KCa channels displayed noted colocalization in the merged images (Merged, yellow) for CAV-1 (green) and SK3 (red) (F), for CAV-1 (red) and BKα (green) (G), and for CAV-1 (green) and IK1 (red) (H). Areas of more diffuse localization were also apparent, especially for BKα and IK1 (see merged images in G and H). Immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: Anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600, anti-SK1 (Alomone, cat. no. APC-039), anti-SK3 (Alomone, cat. no. APC-025-ATTO-594), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti- TRPV4-ATTO-550 (Alomone, cat. no. ACC-034-AO) were used.

Techniques: Immunofluorescence, Immunostaining