anti trpv4 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti trpv4 antibody
    Effects of hypotonicity on the distribution of <t>TRPV4</t> in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Anti Trpv4 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 33 article reviews
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    anti trpv4 antibody - by Bioz Stars, 2022-10
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    Images

    1) Product Images from "Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat"

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2012.e32

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Figure Legend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Techniques Used: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.
    Figure Legend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Techniques Used: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P
    Figure Legend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Techniques Used: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation

    2) Product Images from "Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C *"

    Article Title: Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.466797

    Acute activation of the PKA signaling cascade promotes apical TRPV4 translocation. A , distribution of averaged relative fluorescent signals representing TRPV4 localization along a line on z -axis in individual cells from distal nephrons similar to that
    Figure Legend Snippet: Acute activation of the PKA signaling cascade promotes apical TRPV4 translocation. A , distribution of averaged relative fluorescent signals representing TRPV4 localization along a line on z -axis in individual cells from distal nephrons similar to that

    Techniques Used: Activation Assay, Translocation Assay

    Regulation of mechanosensitive [Ca 2+ ] i responses by PKA and PKC cascades occurs in a TRPV4-dependent manner. A , average time course of changes in [Ca 2+ ] i levels in response to a 10-fold elevation in flow over the apical surface ( gray bars ) for individual
    Figure Legend Snippet: Regulation of mechanosensitive [Ca 2+ ] i responses by PKA and PKC cascades occurs in a TRPV4-dependent manner. A , average time course of changes in [Ca 2+ ] i levels in response to a 10-fold elevation in flow over the apical surface ( gray bars ) for individual

    Techniques Used: Flow Cytometry

    Distinct effects of PKC- and PKA-dependent signaling cascades on subcellular TRPV4 localization in distal nephron cells. Shown are representative confocal plane micrographs (axes are shown) and corresponding cross-sections (indicated by arrows ) demonstrating
    Figure Legend Snippet: Distinct effects of PKC- and PKA-dependent signaling cascades on subcellular TRPV4 localization in distal nephron cells. Shown are representative confocal plane micrographs (axes are shown) and corresponding cross-sections (indicated by arrows ) demonstrating

    Techniques Used:

    3) Product Images from "Differential Localizations of the Transient Receptor Potential Channels TRPV4 and TRPV1 in the Mouse Urinary Bladder"

    Article Title: Differential Localizations of the Transient Receptor Potential Channels TRPV4 and TRPV1 in the Mouse Urinary Bladder

    Journal:

    doi: 10.1369/jhc.2008.951962

    Immunoelectron microscopic analyses of TRPV4 in the mouse urothelium. ( A ) Gold particles, which represented the intracellular epitopes recognized by the TRPV4 polyclonal antibody, were sparsely and intracellularly located near the basal urothelial cell
    Figure Legend Snippet: Immunoelectron microscopic analyses of TRPV4 in the mouse urothelium. ( A ) Gold particles, which represented the intracellular epitopes recognized by the TRPV4 polyclonal antibody, were sparsely and intracellularly located near the basal urothelial cell

    Techniques Used:

    Expression of TRPV4 and TRPV1 transcripts in the mouse urothelium and dorsal root ganglion (DRG) neurons by ISH. ( A,B ) Strong expression of TRPV4 transcripts was observed in the mouse urothelia at low ( A ) and high ( B ) magnifications when the TRPV4 antisense
    Figure Legend Snippet: Expression of TRPV4 and TRPV1 transcripts in the mouse urothelium and dorsal root ganglion (DRG) neurons by ISH. ( A,B ) Strong expression of TRPV4 transcripts was observed in the mouse urothelia at low ( A ) and high ( B ) magnifications when the TRPV4 antisense

    Techniques Used: Expressing, In Situ Hybridization

    Immunoblotting and IHC analyses of TRPV4 in the mouse urothelium. ( A ) Detection of TRPV4 protein (arrowhead) in total extracts of TRPV4-expressing or non-expressing HEK293T cells (Lanes 1 and 2, respectively), and detection of TRPV4 protein in the bladder
    Figure Legend Snippet: Immunoblotting and IHC analyses of TRPV4 in the mouse urothelium. ( A ) Detection of TRPV4 protein (arrowhead) in total extracts of TRPV4-expressing or non-expressing HEK293T cells (Lanes 1 and 2, respectively), and detection of TRPV4 protein in the bladder

    Techniques Used: Immunohistochemistry, Expressing

    4) Product Images from "Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat"

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2012.e32

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Figure Legend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Techniques Used: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.
    Figure Legend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Techniques Used: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P
    Figure Legend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Techniques Used: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation

    5) Product Images from "Identification of TRPV4 as a novel target in invasiveness of colorectal cancer"

    Article Title: Identification of TRPV4 as a novel target in invasiveness of colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-021-08970-7

    TRPV4 promotes colon cancer cell migration and invasion through regulating epithelial-mesenchymal transition process. A Western blot analysis of TRPV4 showing increased TRPV4 levels in HT-29 cells transfected with a TRPV4-overexpression construct. B TRPV4 mRNA levels in HT-29 cells transfected with a TRPV4-overexpression construct. C and D representative images and summary data of migration and invasion assay in HT-29 cells transfected with a TRPV4-overexpression construct, scale bar: 50 μm. E and F RNA levels of the epithelial-mesenchymal transition (EMT) markers in SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047. G Western blot analysis for EMT markers showing a decreased EMT process in SW620 cells transfected with siCTL, siTRPV4#1, and siTRPV4#2 or treated with Vehicle or HC-067047. H Western blot analysis for EMT markers showing an increased EMT process in HT-29 cells transfected with a TRPV4-overexpression construct. I Summary data for A. J Summary data for G. K Summary data for H. Values represent the mean ± SEM, #, p
    Figure Legend Snippet: TRPV4 promotes colon cancer cell migration and invasion through regulating epithelial-mesenchymal transition process. A Western blot analysis of TRPV4 showing increased TRPV4 levels in HT-29 cells transfected with a TRPV4-overexpression construct. B TRPV4 mRNA levels in HT-29 cells transfected with a TRPV4-overexpression construct. C and D representative images and summary data of migration and invasion assay in HT-29 cells transfected with a TRPV4-overexpression construct, scale bar: 50 μm. E and F RNA levels of the epithelial-mesenchymal transition (EMT) markers in SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047. G Western blot analysis for EMT markers showing a decreased EMT process in SW620 cells transfected with siCTL, siTRPV4#1, and siTRPV4#2 or treated with Vehicle or HC-067047. H Western blot analysis for EMT markers showing an increased EMT process in HT-29 cells transfected with a TRPV4-overexpression construct. I Summary data for A. J Summary data for G. K Summary data for H. Values represent the mean ± SEM, #, p

    Techniques Used: Migration, Western Blot, Transfection, Over Expression, Construct, Invasion Assay

    TRPV4 expression is correlated with tumor metastasis in human colon cancer specimens. A and B representative images and summary data of immunohistochemical staining of TRPV4 and ZEB1 in patients with or without lymph node metastasis (N0 n = 59, N1–2 n = 47) Scale bar: 50 μm. C Pearson correction of TRPV4 expression with ZEB1 ( n = 106), data were analyzed using the Pearson correlation test. D Signaling connections involved in the TRPV4-modulated epithelial-mesenchymal transition (EMT) process in human colon cancer
    Figure Legend Snippet: TRPV4 expression is correlated with tumor metastasis in human colon cancer specimens. A and B representative images and summary data of immunohistochemical staining of TRPV4 and ZEB1 in patients with or without lymph node metastasis (N0 n = 59, N1–2 n = 47) Scale bar: 50 μm. C Pearson correction of TRPV4 expression with ZEB1 ( n = 106), data were analyzed using the Pearson correlation test. D Signaling connections involved in the TRPV4-modulated epithelial-mesenchymal transition (EMT) process in human colon cancer

    Techniques Used: Expressing, Immunohistochemistry, Staining

    TRPV4 increases ZEB1 expression to enhance epithelial-mesenchymal transition. A and B Western blot analysis for p-AKT, AKT, ZEB1 and ATCB in HCT-116 and SW620 cells transfected with siCTL and siTRPV4#1 or treated with Vehicle or HC-067047. C and D representative images and summary data of Western blot demonstrating the effects of ZEB1 siRNA or AKT siRNA on the EMT process induced by TRPV4 overexpression in HT-29 cells. E and F representative images and summary data from migration and invasion assay demonstrating the effects of ZEB1 siRNA on TRPV4 overexpression induced cell motility, scale bar: 50 μm. G and H representative images and summary data from migration and invasion assay demonstrating the effects of AKT siRNA on TRPV4 overexpression induced cell motility, scale bar: 50 μm. Values represent the mean ± SEM, #, p
    Figure Legend Snippet: TRPV4 increases ZEB1 expression to enhance epithelial-mesenchymal transition. A and B Western blot analysis for p-AKT, AKT, ZEB1 and ATCB in HCT-116 and SW620 cells transfected with siCTL and siTRPV4#1 or treated with Vehicle or HC-067047. C and D representative images and summary data of Western blot demonstrating the effects of ZEB1 siRNA or AKT siRNA on the EMT process induced by TRPV4 overexpression in HT-29 cells. E and F representative images and summary data from migration and invasion assay demonstrating the effects of ZEB1 siRNA on TRPV4 overexpression induced cell motility, scale bar: 50 μm. G and H representative images and summary data from migration and invasion assay demonstrating the effects of AKT siRNA on TRPV4 overexpression induced cell motility, scale bar: 50 μm. Values represent the mean ± SEM, #, p

    Techniques Used: Expressing, Western Blot, Transfection, Over Expression, Migration, Invasion Assay

    Inhibition of TRPV4 suppresses colon cancer cell migration and invasion. A and B representative images and summary data of the migration assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047, scale bar: 50 μm. C and D representative images and summary data of the wound healing assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047. E and F Representative images and summary data from invasion assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047, scale bar: 50 μm. Values represent the mean ± SEM, #, p
    Figure Legend Snippet: Inhibition of TRPV4 suppresses colon cancer cell migration and invasion. A and B representative images and summary data of the migration assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047, scale bar: 50 μm. C and D representative images and summary data of the wound healing assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047. E and F Representative images and summary data from invasion assay in HCT-116 cells and SW620 cells transfected with siCTL, siTRPV4#1 and siTRPV4#2 or treated with Vehicle or HC-067047, scale bar: 50 μm. Values represent the mean ± SEM, #, p

    Techniques Used: Inhibition, Migration, Transfection, Wound Healing Assay, Invasion Assay

    6) Product Images from "TRPV4 calcium entry and surface expression attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells"

    Article Title: TRPV4 calcium entry and surface expression attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells

    Journal: Physiological Reports

    doi: 10.1002/phy2.121

    Western blot analysis of surface TRPV4 protein expression in RPMVEC under baseline conditions and after incubation with ML-7. * P
    Figure Legend Snippet: Western blot analysis of surface TRPV4 protein expression in RPMVEC under baseline conditions and after incubation with ML-7. * P

    Techniques Used: Western Blot, Expressing, Incubation

    Confocal fluorescent micrographs showing localization of focal adhesion kinase (green) and TRPV4 channels (red) in basilar region of RPMVEC. Cell nuclei are shown in blue.
    Figure Legend Snippet: Confocal fluorescent micrographs showing localization of focal adhesion kinase (green) and TRPV4 channels (red) in basilar region of RPMVEC. Cell nuclei are shown in blue.

    Techniques Used:

    Western blot of surface expression of TRPV4 on RPMVEC monolayers under baseline conditions and after treatment with dynasore. Blots were spiced from the same MW band (vertical black line) and equal loading of samples was confirmed by Ponceau S staining.
    Figure Legend Snippet: Western blot of surface expression of TRPV4 on RPMVEC monolayers under baseline conditions and after treatment with dynasore. Blots were spiced from the same MW band (vertical black line) and equal loading of samples was confirmed by Ponceau S staining.

    Techniques Used: Western Blot, Expressing, Staining

    7) Product Images from "Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat"

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2012.e32

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Figure Legend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Techniques Used: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.
    Figure Legend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Techniques Used: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P
    Figure Legend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Techniques Used: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation

    8) Product Images from "Effects of Body Temperature on Neural Activity in the Hippocampus: Regulation of Resting Membrane Potentials by Transient Receptor Potential Vanilloid 4"

    Article Title: Effects of Body Temperature on Neural Activity in the Hippocampus: Regulation of Resting Membrane Potentials by Transient Receptor Potential Vanilloid 4

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4284-06.2007

    TRPV4-EGFP localized at postsynapses in matured hippocampal neurons. A , Synapsin-I (green) and PSD-95 (red) were colocalized in dissociated hippocampal neurons from WT mice at 21 DIV (yellow in a merged image). B , Exogenous TRPV4 (green) was localized in the soma (except nucleus) and dendrites of cultured WT hippocampal neurons at 21 DIV (2 d after transfection of EGFP-fused TRPV4, TRPV4-EGFP). C , Exogenous TRPV4 (green) was colocalized with synapsin-I (red) and phalloidin (blue) in spines of cultured WT hippocampal neurons. Scale bars, 50 μm.
    Figure Legend Snippet: TRPV4-EGFP localized at postsynapses in matured hippocampal neurons. A , Synapsin-I (green) and PSD-95 (red) were colocalized in dissociated hippocampal neurons from WT mice at 21 DIV (yellow in a merged image). B , Exogenous TRPV4 (green) was localized in the soma (except nucleus) and dendrites of cultured WT hippocampal neurons at 21 DIV (2 d after transfection of EGFP-fused TRPV4, TRPV4-EGFP). C , Exogenous TRPV4 (green) was colocalized with synapsin-I (red) and phalloidin (blue) in spines of cultured WT hippocampal neurons. Scale bars, 50 μm.

    Techniques Used: Mouse Assay, Cell Culture, Transfection

    A proposed model for TRPV4 involvement in the regulation of RMPs in hippocampal pyramidal neurons. RMPs were not significantly different between wild-type and TRPV4KO neurons at approximately –62 mV at 25°C. RMPs were significantly depolarized to a value close to the threshold for NMDA receptor activation in wild-type neurons compared with TRPV4KO at 37°C, leading to more excitability.
    Figure Legend Snippet: A proposed model for TRPV4 involvement in the regulation of RMPs in hippocampal pyramidal neurons. RMPs were not significantly different between wild-type and TRPV4KO neurons at approximately –62 mV at 25°C. RMPs were significantly depolarized to a value close to the threshold for NMDA receptor activation in wild-type neurons compared with TRPV4KO at 37°C, leading to more excitability.

    Techniques Used: Activation Assay

    TRPV4 expression is responsible for neural excitability through changes in RMPs in hippocampal pyramidal neurons. A , Average RMP (with SEM) at 25 and 37°C in WT and TRPV4KO neurons. * p
    Figure Legend Snippet: TRPV4 expression is responsible for neural excitability through changes in RMPs in hippocampal pyramidal neurons. A , Average RMP (with SEM) at 25 and 37°C in WT and TRPV4KO neurons. * p

    Techniques Used: Expressing

    9) Product Images from "Transient receptor potential vanilloid 4 as a regulator of induced pluripotent stem cell chondrogenesis"

    Article Title: Transient receptor potential vanilloid 4 as a regulator of induced pluripotent stem cell chondrogenesis

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.3440

    TRPV4-mediated Ca 2+ signaling. A, GFP-sorted induced pluripotent stem cell (iPSC)-derived chondroprogenitor cell response to TRPV4-mediated Ca 2+ signaling with GSK1016790A (GSK101) treatment measured by confocal cell traces, as compared to undifferentiated iPSCs or primary murine articular chondrocytes (AC) (N = 3 experiments, n = 78-161 cell traces per group; groups not sharing the same letter are statistically different from one another by Chi-square test, P
    Figure Legend Snippet: TRPV4-mediated Ca 2+ signaling. A, GFP-sorted induced pluripotent stem cell (iPSC)-derived chondroprogenitor cell response to TRPV4-mediated Ca 2+ signaling with GSK1016790A (GSK101) treatment measured by confocal cell traces, as compared to undifferentiated iPSCs or primary murine articular chondrocytes (AC) (N = 3 experiments, n = 78-161 cell traces per group; groups not sharing the same letter are statistically different from one another by Chi-square test, P

    Techniques Used: Derivative Assay

    Trpv4 expression within chondrogenic cell population identified by Col2a1-GFP reporter. Induced pluripotent stem cells (iPSCs) underwent chondrogenesis for 15 days and were sorted into GFP+ and GFP− populations based on a Col2a1-GFP reporter before quantitative reverse transcription polymerase chain reaction (qRT-PCR) gene expression analysis for pluripotency gene Nanog , chondrogenic markers Sox9, Acan , and Col2a1 , and Trpv4 . Controls: undifferentiated iPSCs and isolated primary murine articular chondrocytes (AC). Mean ± SEM, n = 5, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P
    Figure Legend Snippet: Trpv4 expression within chondrogenic cell population identified by Col2a1-GFP reporter. Induced pluripotent stem cells (iPSCs) underwent chondrogenesis for 15 days and were sorted into GFP+ and GFP− populations based on a Col2a1-GFP reporter before quantitative reverse transcription polymerase chain reaction (qRT-PCR) gene expression analysis for pluripotency gene Nanog , chondrogenic markers Sox9, Acan , and Col2a1 , and Trpv4 . Controls: undifferentiated iPSCs and isolated primary murine articular chondrocytes (AC). Mean ± SEM, n = 5, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

    Cell proliferation, sulfated glycosaminoglycan (sGAG) production, and chondrogenic gene expression in induced pluripotent stem cell (iPSC)-derived chondroprogenitors after transient activation of TRPV4. A, GFP+ and GFP− cells were formed into micromasses and underwent chondrogenesis for 7 days before being treated with GSK1016790A (GSK101) or GSK101+GSK205 for 1 hour per day, in the absence of chondrogenic growth factors. B, Day 21 biochemical analysis for DNA, sGAG, and collagen content showed that activation of TRPV4 with GSK101 increased DNA content, sGAG/DNA ratio in GFP+ groups only. Total collagen/DNA was not affected. C, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) at day 21 showed significantly increased Sox9 and Acan expression in GFP+ cells in response to GSK101 treatment compared to controls conditions. Col2a1 expression was not significantly altered in either cell population in response to TRPV4 activation. Mean ± SEM, n = 5-6 for qPCR, n = 5 for biochemical measurements, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P
    Figure Legend Snippet: Cell proliferation, sulfated glycosaminoglycan (sGAG) production, and chondrogenic gene expression in induced pluripotent stem cell (iPSC)-derived chondroprogenitors after transient activation of TRPV4. A, GFP+ and GFP− cells were formed into micromasses and underwent chondrogenesis for 7 days before being treated with GSK1016790A (GSK101) or GSK101+GSK205 for 1 hour per day, in the absence of chondrogenic growth factors. B, Day 21 biochemical analysis for DNA, sGAG, and collagen content showed that activation of TRPV4 with GSK101 increased DNA content, sGAG/DNA ratio in GFP+ groups only. Total collagen/DNA was not affected. C, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) at day 21 showed significantly increased Sox9 and Acan expression in GFP+ cells in response to GSK101 treatment compared to controls conditions. Col2a1 expression was not significantly altered in either cell population in response to TRPV4 activation. Mean ± SEM, n = 5-6 for qPCR, n = 5 for biochemical measurements, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P

    Techniques Used: Expressing, Derivative Assay, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Trpv4 expression during chondrogenesis of induced pluripotent stem cells (iPSCs). A, Alcian blue staining of iPSCs undergoing chondrogenic differentiation over 21 days. B, Gene expression for pluripotency genes Nanog and Sox2 , mesenchymal marker Vim , and chondrogenic markers Sox9, Acan , and Col2a1 , and Trpv4 . Mean ± SEM, n = 5 per group, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P
    Figure Legend Snippet: Trpv4 expression during chondrogenesis of induced pluripotent stem cells (iPSCs). A, Alcian blue staining of iPSCs undergoing chondrogenic differentiation over 21 days. B, Gene expression for pluripotency genes Nanog and Sox2 , mesenchymal marker Vim , and chondrogenic markers Sox9, Acan , and Col2a1 , and Trpv4 . Mean ± SEM, n = 5 per group, groups not sharing the same letter are statistically different from one another (analysis of variance with Tukey's post hoc, P

    Techniques Used: Expressing, Staining, Marker

    10) Product Images from "TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats"

    Article Title: TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats

    Journal: JCI Insight

    doi: 10.1172/jci.insight.137528

    Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P
    Figure Legend Snippet: Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P

    Techniques Used: Expressing, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P
    Figure Legend Snippet: TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, BrdU Staining

    The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P
    Figure Legend Snippet: The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P

    Techniques Used: Isolation, Activation Assay

    Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P
    Figure Legend Snippet: Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P

    Techniques Used: Isolation

    Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P
    Figure Legend Snippet: Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P

    Techniques Used: Western Blot

    Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P
    Figure Legend Snippet: Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Negative Control

    11) Product Images from "Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat"

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2012.e32

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Figure Legend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Techniques Used: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.
    Figure Legend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Techniques Used: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P
    Figure Legend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Techniques Used: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation

    12) Product Images from "Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion"

    Article Title: Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion

    Journal: eLife

    doi: 10.7554/eLife.78840

    siRNA knockdown supports TRPV4 functional expression in BeWo cells. ( A ) Representative Ca 2+ imaging in BeWo cells transfected with scrambled siRNA and TRPV4 siRNAs. Calbryte 590 was used to monitor cytosolic Ca 2+ dynamics. All fluorescence images are the representatives of at least three biological replicates. ( B ) Quantification of GSK101-induced Ca 2+ influx in BeWo cells transfected with scrambled siRNA (n=50), TRPV4 siRNA2 (n=55), or siRNA4 (n=51) from at least three biological replicates. Values represent mean ± SEM and statistics were done using two-way ANOVA followed by Tukey’s test (**: p
    Figure Legend Snippet: siRNA knockdown supports TRPV4 functional expression in BeWo cells. ( A ) Representative Ca 2+ imaging in BeWo cells transfected with scrambled siRNA and TRPV4 siRNAs. Calbryte 590 was used to monitor cytosolic Ca 2+ dynamics. All fluorescence images are the representatives of at least three biological replicates. ( B ) Quantification of GSK101-induced Ca 2+ influx in BeWo cells transfected with scrambled siRNA (n=50), TRPV4 siRNA2 (n=55), or siRNA4 (n=51) from at least three biological replicates. Values represent mean ± SEM and statistics were done using two-way ANOVA followed by Tukey’s test (**: p

    Techniques Used: Functional Assay, Expressing, Imaging, Transfection, Fluorescence

    Ca 2+ influx through TRPV4 activates TMEM16F scramblase (left) and siRNA knockdown of TRPV4 (right) abolishes GSK101-induced ca 2+ influx and subsequent TMEM16F CaPLSase activation.
    Figure Legend Snippet: Ca 2+ influx through TRPV4 activates TMEM16F scramblase (left) and siRNA knockdown of TRPV4 (right) abolishes GSK101-induced ca 2+ influx and subsequent TMEM16F CaPLSase activation.

    Techniques Used: Activation Assay

    TRPV4 knockout (KO) placentas do not show obvious defects in trophoblast syncytialization in mice. ( A, B ) Representative images of placentas from wild-type (WT) ( A ) and TRPV4 KO ( B ) mice at E18.5. ( C ) Anatomical diagram of fetomaternal exchange in mouse placentas. ( D, E ) MCT1 and MCT4 immunofluorescence staining of the TRPV4 WT ( D ) and KO ( E ) placentas at E18.5. MCT1 (red) specifically stains the SynT-1 layer that faces maternal blood sinuses, while MCT4 (green) specifically stains the SynT-2 layer that encloses fetal blood vessels. Panels ( i–iv ) at the bottom show enlarged views of the placentas on top. All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: TRPV4 knockout (KO) placentas do not show obvious defects in trophoblast syncytialization in mice. ( A, B ) Representative images of placentas from wild-type (WT) ( A ) and TRPV4 KO ( B ) mice at E18.5. ( C ) Anatomical diagram of fetomaternal exchange in mouse placentas. ( D, E ) MCT1 and MCT4 immunofluorescence staining of the TRPV4 WT ( D ) and KO ( E ) placentas at E18.5. MCT1 (red) specifically stains the SynT-1 layer that faces maternal blood sinuses, while MCT4 (green) specifically stains the SynT-2 layer that encloses fetal blood vessels. Panels ( i–iv ) at the bottom show enlarged views of the placentas on top. All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Knock-Out, Mouse Assay, Immunofluorescence, Staining, Fluorescence

    Immunofluorescence of TRPV4 in BeWo cells transfected with TRPV4 siRNAs. Representative immunofluorescence of TRPV4 (red) in scrambled control siRNA and TRPV4 siRNA knockdown BeWo cells. The nuclei were stained with Hoechst (blue). The plasma membrane was labeled with WGA dye (green). All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: Immunofluorescence of TRPV4 in BeWo cells transfected with TRPV4 siRNAs. Representative immunofluorescence of TRPV4 (red) in scrambled control siRNA and TRPV4 siRNA knockdown BeWo cells. The nuclei were stained with Hoechst (blue). The plasma membrane was labeled with WGA dye (green). All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Immunofluorescence, Transfection, Staining, Labeling, Whole Genome Amplification, Fluorescence

    Validation of the TRPV4 antibody for immunofluorescence. ( A, B ) Validation of the TRPV4 antibody in HEK293T cells heterologously expressing a Flag-tagged TRPV4 plasmid. Immunofluorescence of TRPV4 (anti-TRPV4, green) and Flag tag (anti-Flag, red) in HEK293T cells transfected with a Flag-tagged TRPV4 plasmid ( A ) and non-transfected control ( B ). DAPI-stained nuclei are shown in blue. ( C ) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue) and a plasma membrane marker, FM1-43 dye, is shown in red. All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: Validation of the TRPV4 antibody for immunofluorescence. ( A, B ) Validation of the TRPV4 antibody in HEK293T cells heterologously expressing a Flag-tagged TRPV4 plasmid. Immunofluorescence of TRPV4 (anti-TRPV4, green) and Flag tag (anti-Flag, red) in HEK293T cells transfected with a Flag-tagged TRPV4 plasmid ( A ) and non-transfected control ( B ). DAPI-stained nuclei are shown in blue. ( C ) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue) and a plasma membrane marker, FM1-43 dye, is shown in red. All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Immunofluorescence, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Staining, Marker, Fluorescence

    Pharmacological inhibition of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation in BeWo cells. ( A ) Representative images Ca 2+ and PS exposure in BeWo cells treated with 20 nM TRPV4 agonist GSK101 and by 500 nM TRPV4 antagonist GSK219. Ca 2+ dye (Calbryte 488) and fluorescently tagged AnV proteins (AnV-CF594) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. ( B, C ) Time course of intracellular Ca 2+ ( B ) and PS exposure ( C ) in BeWo cells treated with 20 nM GSK101 and by 500 nM GSK219. n=9. All fluorescence images are the representatives of at least three biological replicates. AnV, Annexin V.
    Figure Legend Snippet: Pharmacological inhibition of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation in BeWo cells. ( A ) Representative images Ca 2+ and PS exposure in BeWo cells treated with 20 nM TRPV4 agonist GSK101 and by 500 nM TRPV4 antagonist GSK219. Ca 2+ dye (Calbryte 488) and fluorescently tagged AnV proteins (AnV-CF594) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. ( B, C ) Time course of intracellular Ca 2+ ( B ) and PS exposure ( C ) in BeWo cells treated with 20 nM GSK101 and by 500 nM GSK219. n=9. All fluorescence images are the representatives of at least three biological replicates. AnV, Annexin V.

    Techniques Used: Inhibition, Activation Assay, Fluorescence

    Simultaneous imaging of Ca 2+ increase and phospholipid scrambling in TMEM16F knockout (KO) BeWo cells in response to low concentration GSK101. ( A ) Stimulation of TRPV4 with low concentration of GSK101 (0.2 nM) triggers transient Ca 2+ increase without spatiotemporal PS exposure in TMEM16F KO BeWo cells. Ca 2+ dye (Calbryte 594) and fluorescently tagged AnV proteins (AnV-CF488) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( B ) The dynamics of Ca 2+ (black) and AnV signal (red) of a TMEM16F KO BeWo cell in ( A ) (*). AnV, Annexin V.
    Figure Legend Snippet: Simultaneous imaging of Ca 2+ increase and phospholipid scrambling in TMEM16F knockout (KO) BeWo cells in response to low concentration GSK101. ( A ) Stimulation of TRPV4 with low concentration of GSK101 (0.2 nM) triggers transient Ca 2+ increase without spatiotemporal PS exposure in TMEM16F KO BeWo cells. Ca 2+ dye (Calbryte 594) and fluorescently tagged AnV proteins (AnV-CF488) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( B ) The dynamics of Ca 2+ (black) and AnV signal (red) of a TMEM16F KO BeWo cell in ( A ) (*). AnV, Annexin V.

    Techniques Used: Imaging, Knock-Out, Concentration Assay, Fluorescence

    Lack of TRPV4-TMEM16F coupling in BeWo trophoblast cells in the absence of GSK101. ( A ) Outside-out patch recording of BeWo cells in the absence of GSK101. (1) 0 extracellular Ca 2+ ; (2) 2.5 mM extracellular Ca 2+ . 0.2 mM EGTA was included in the pipette. ( B ) Comparison of outside-out patch current with ( Figure 5B , #2, with 2.5 mM Ca 2+ ) and without GSK101 after 1 s of pre-pulse. ( C ) Comparison of outside-out patch current with ( Figure 5B , #1, with 0 Ca 2+ ) and without GSK101 after 1 s of pre-pulse. Values represent mean ± SEM and statistics were done using Student’s t-test (n=5, ****: p
    Figure Legend Snippet: Lack of TRPV4-TMEM16F coupling in BeWo trophoblast cells in the absence of GSK101. ( A ) Outside-out patch recording of BeWo cells in the absence of GSK101. (1) 0 extracellular Ca 2+ ; (2) 2.5 mM extracellular Ca 2+ . 0.2 mM EGTA was included in the pipette. ( B ) Comparison of outside-out patch current with ( Figure 5B , #2, with 2.5 mM Ca 2+ ) and without GSK101 after 1 s of pre-pulse. ( C ) Comparison of outside-out patch current with ( Figure 5B , #1, with 0 Ca 2+ ) and without GSK101 after 1 s of pre-pulse. Values represent mean ± SEM and statistics were done using Student’s t-test (n=5, ****: p

    Techniques Used: Transferring

    TRPV4 and TMEM16F are spatially close to each other on BeWo cell membrane.
    Figure Legend Snippet: TRPV4 and TMEM16F are spatially close to each other on BeWo cell membrane.

    Techniques Used:

    4α-phorbol-12, 13-didecanoate (4α-PDD, 20 μM), a TRPV4 agonist, triggers intracellular Ca 2+ elevation in BeWo cells. ( A ) Ca 2+ dye (Calbryte 520, green) was used to monitor the dynamics of intracellular Ca 2+ . All fluorescence images are the representatives of at least three biological replicates. ( B ) Time course of 4α-PDD triggered Ca 2+ influx in BeWo cells (n=7).
    Figure Legend Snippet: 4α-phorbol-12, 13-didecanoate (4α-PDD, 20 μM), a TRPV4 agonist, triggers intracellular Ca 2+ elevation in BeWo cells. ( A ) Ca 2+ dye (Calbryte 520, green) was used to monitor the dynamics of intracellular Ca 2+ . All fluorescence images are the representatives of at least three biological replicates. ( B ) Time course of 4α-PDD triggered Ca 2+ influx in BeWo cells (n=7).

    Techniques Used: Fluorescence

    TMEM16F CaPLSase and channel activity in BeWo cells is not affected by TRPV4 knockdown. ( A ) Representative images showing 5 μM ionomycin triggers similar levels of PS exposure in scrambled, TPRV4 siRNA2, and siRNA4 knockdown BeWo cells. ( B ) Quantification of ionomycin-induced AnV intensities increase in BeWo cells transfected with scrambled siRNA (n=25), TRPV4 siRNA2 (n=25), or siRNA4 (n=27). All fluorescence images are the representatives of at least three biological replicates. ( C ) Representative TMEM16F current traces from control siRNA and the TRPV4 siRNAs treated BeWo cells recorded with whole-cell patch clamp or patch clamp-lipid scrambling fluorometry (PCLSF). 1000 μM Ca 2+ was included in the pipette solution, and the current was elicited by a voltage step protocol from –100 to +160 mV with holding potential at –60 mV. ( D ) I-V relation of TMEM16F current recorded from the control siRNA and the TRPV4 siRNAs treated BeWo cells. ( E ) Statistics of the TMEM16F current density at +160 mV shown in ( D ). Student’s t-test (ns: not significant, n=7 for control siRNA, n=8 for TRPV4 siRNA2, and n=7 for TRPV4 siRNA4). ( F ) Representative lipid scrambling activities recorded using PCLSF to simultaneously record TMEM16F channel and lipid scramblase activities. The cells are the same ones shown in ( C ). n=3. AnV, Annexin V.
    Figure Legend Snippet: TMEM16F CaPLSase and channel activity in BeWo cells is not affected by TRPV4 knockdown. ( A ) Representative images showing 5 μM ionomycin triggers similar levels of PS exposure in scrambled, TPRV4 siRNA2, and siRNA4 knockdown BeWo cells. ( B ) Quantification of ionomycin-induced AnV intensities increase in BeWo cells transfected with scrambled siRNA (n=25), TRPV4 siRNA2 (n=25), or siRNA4 (n=27). All fluorescence images are the representatives of at least three biological replicates. ( C ) Representative TMEM16F current traces from control siRNA and the TRPV4 siRNAs treated BeWo cells recorded with whole-cell patch clamp or patch clamp-lipid scrambling fluorometry (PCLSF). 1000 μM Ca 2+ was included in the pipette solution, and the current was elicited by a voltage step protocol from –100 to +160 mV with holding potential at –60 mV. ( D ) I-V relation of TMEM16F current recorded from the control siRNA and the TRPV4 siRNAs treated BeWo cells. ( E ) Statistics of the TMEM16F current density at +160 mV shown in ( D ). Student’s t-test (ns: not significant, n=7 for control siRNA, n=8 for TRPV4 siRNA2, and n=7 for TRPV4 siRNA4). ( F ) Representative lipid scrambling activities recorded using PCLSF to simultaneously record TMEM16F channel and lipid scramblase activities. The cells are the same ones shown in ( C ). n=3. AnV, Annexin V.

    Techniques Used: Activity Assay, Transfection, Fluorescence, Patch Clamp, Transferring

    13) Product Images from "TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats"

    Article Title: TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats

    Journal: JCI Insight

    doi: 10.1172/jci.insight.137528

    Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P
    Figure Legend Snippet: Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P

    Techniques Used: Expressing, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P
    Figure Legend Snippet: TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, BrdU Staining

    The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P
    Figure Legend Snippet: The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P

    Techniques Used: Isolation, Activation Assay

    Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P
    Figure Legend Snippet: Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P

    Techniques Used: Isolation

    Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P
    Figure Legend Snippet: Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P

    Techniques Used: Western Blot

    Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P
    Figure Legend Snippet: Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Negative Control

    14) Product Images from "Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion"

    Article Title: Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion

    Journal: eLife

    doi: 10.7554/eLife.78840

    siRNA knockdown supports TRPV4 functional expression in BeWo cells. ( A ) Representative Ca 2+ imaging in BeWo cells transfected with scrambled siRNA and TRPV4 siRNAs. Calbryte 590 was used to monitor cytosolic Ca 2+ dynamics. All fluorescence images are the representatives of at least three biological replicates. ( B ) Quantification of GSK101-induced Ca 2+ influx in BeWo cells transfected with scrambled siRNA (n=50), TRPV4 siRNA2 (n=55), or siRNA4 (n=51) from at least three biological replicates. Values represent mean ± SEM and statistics were done using two-way ANOVA followed by Tukey’s test (**: p
    Figure Legend Snippet: siRNA knockdown supports TRPV4 functional expression in BeWo cells. ( A ) Representative Ca 2+ imaging in BeWo cells transfected with scrambled siRNA and TRPV4 siRNAs. Calbryte 590 was used to monitor cytosolic Ca 2+ dynamics. All fluorescence images are the representatives of at least three biological replicates. ( B ) Quantification of GSK101-induced Ca 2+ influx in BeWo cells transfected with scrambled siRNA (n=50), TRPV4 siRNA2 (n=55), or siRNA4 (n=51) from at least three biological replicates. Values represent mean ± SEM and statistics were done using two-way ANOVA followed by Tukey’s test (**: p

    Techniques Used: Functional Assay, Expressing, Imaging, Transfection, Fluorescence

    Ca 2+ influx through TRPV4 activates TMEM16F scramblase (left) and siRNA knockdown of TRPV4 (right) abolishes GSK101-induced ca 2+ influx and subsequent TMEM16F CaPLSase activation.
    Figure Legend Snippet: Ca 2+ influx through TRPV4 activates TMEM16F scramblase (left) and siRNA knockdown of TRPV4 (right) abolishes GSK101-induced ca 2+ influx and subsequent TMEM16F CaPLSase activation.

    Techniques Used: Activation Assay

    TRPV4 knockout (KO) placentas do not show obvious defects in trophoblast syncytialization in mice. ( A, B ) Representative images of placentas from wild-type (WT) ( A ) and TRPV4 KO ( B ) mice at E18.5. ( C ) Anatomical diagram of fetomaternal exchange in mouse placentas. ( D, E ) MCT1 and MCT4 immunofluorescence staining of the TRPV4 WT ( D ) and KO ( E ) placentas at E18.5. MCT1 (red) specifically stains the SynT-1 layer that faces maternal blood sinuses, while MCT4 (green) specifically stains the SynT-2 layer that encloses fetal blood vessels. Panels ( i–iv ) at the bottom show enlarged views of the placentas on top. All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: TRPV4 knockout (KO) placentas do not show obvious defects in trophoblast syncytialization in mice. ( A, B ) Representative images of placentas from wild-type (WT) ( A ) and TRPV4 KO ( B ) mice at E18.5. ( C ) Anatomical diagram of fetomaternal exchange in mouse placentas. ( D, E ) MCT1 and MCT4 immunofluorescence staining of the TRPV4 WT ( D ) and KO ( E ) placentas at E18.5. MCT1 (red) specifically stains the SynT-1 layer that faces maternal blood sinuses, while MCT4 (green) specifically stains the SynT-2 layer that encloses fetal blood vessels. Panels ( i–iv ) at the bottom show enlarged views of the placentas on top. All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Knock-Out, Mouse Assay, Immunofluorescence, Staining, Fluorescence

    Immunofluorescence of TRPV4 in BeWo cells transfected with TRPV4 siRNAs. Representative immunofluorescence of TRPV4 (red) in scrambled control siRNA and TRPV4 siRNA knockdown BeWo cells. The nuclei were stained with Hoechst (blue). The plasma membrane was labeled with WGA dye (green). All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: Immunofluorescence of TRPV4 in BeWo cells transfected with TRPV4 siRNAs. Representative immunofluorescence of TRPV4 (red) in scrambled control siRNA and TRPV4 siRNA knockdown BeWo cells. The nuclei were stained with Hoechst (blue). The plasma membrane was labeled with WGA dye (green). All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Immunofluorescence, Transfection, Staining, Labeling, Whole Genome Amplification, Fluorescence

    Validation of the TRPV4 antibody for immunofluorescence. ( A, B ) Validation of the TRPV4 antibody in HEK293T cells heterologously expressing a Flag-tagged TRPV4 plasmid. Immunofluorescence of TRPV4 (anti-TRPV4, green) and Flag tag (anti-Flag, red) in HEK293T cells transfected with a Flag-tagged TRPV4 plasmid ( A ) and non-transfected control ( B ). DAPI-stained nuclei are shown in blue. ( C ) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue) and a plasma membrane marker, FM1-43 dye, is shown in red. All fluorescence images are the representatives of at least three biological replicates.
    Figure Legend Snippet: Validation of the TRPV4 antibody for immunofluorescence. ( A, B ) Validation of the TRPV4 antibody in HEK293T cells heterologously expressing a Flag-tagged TRPV4 plasmid. Immunofluorescence of TRPV4 (anti-TRPV4, green) and Flag tag (anti-Flag, red) in HEK293T cells transfected with a Flag-tagged TRPV4 plasmid ( A ) and non-transfected control ( B ). DAPI-stained nuclei are shown in blue. ( C ) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue) and a plasma membrane marker, FM1-43 dye, is shown in red. All fluorescence images are the representatives of at least three biological replicates.

    Techniques Used: Immunofluorescence, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Staining, Marker, Fluorescence

    Pharmacological inhibition of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation in BeWo cells. ( A ) Representative images Ca 2+ and PS exposure in BeWo cells treated with 20 nM TRPV4 agonist GSK101 and by 500 nM TRPV4 antagonist GSK219. Ca 2+ dye (Calbryte 488) and fluorescently tagged AnV proteins (AnV-CF594) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. ( B, C ) Time course of intracellular Ca 2+ ( B ) and PS exposure ( C ) in BeWo cells treated with 20 nM GSK101 and by 500 nM GSK219. n=9. All fluorescence images are the representatives of at least three biological replicates. AnV, Annexin V.
    Figure Legend Snippet: Pharmacological inhibition of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation in BeWo cells. ( A ) Representative images Ca 2+ and PS exposure in BeWo cells treated with 20 nM TRPV4 agonist GSK101 and by 500 nM TRPV4 antagonist GSK219. Ca 2+ dye (Calbryte 488) and fluorescently tagged AnV proteins (AnV-CF594) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. ( B, C ) Time course of intracellular Ca 2+ ( B ) and PS exposure ( C ) in BeWo cells treated with 20 nM GSK101 and by 500 nM GSK219. n=9. All fluorescence images are the representatives of at least three biological replicates. AnV, Annexin V.

    Techniques Used: Inhibition, Activation Assay, Fluorescence

    Simultaneous imaging of Ca 2+ increase and phospholipid scrambling in TMEM16F knockout (KO) BeWo cells in response to low concentration GSK101. ( A ) Stimulation of TRPV4 with low concentration of GSK101 (0.2 nM) triggers transient Ca 2+ increase without spatiotemporal PS exposure in TMEM16F KO BeWo cells. Ca 2+ dye (Calbryte 594) and fluorescently tagged AnV proteins (AnV-CF488) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( B ) The dynamics of Ca 2+ (black) and AnV signal (red) of a TMEM16F KO BeWo cell in ( A ) (*). AnV, Annexin V.
    Figure Legend Snippet: Simultaneous imaging of Ca 2+ increase and phospholipid scrambling in TMEM16F knockout (KO) BeWo cells in response to low concentration GSK101. ( A ) Stimulation of TRPV4 with low concentration of GSK101 (0.2 nM) triggers transient Ca 2+ increase without spatiotemporal PS exposure in TMEM16F KO BeWo cells. Ca 2+ dye (Calbryte 594) and fluorescently tagged AnV proteins (AnV-CF488) were used to measure the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( B ) The dynamics of Ca 2+ (black) and AnV signal (red) of a TMEM16F KO BeWo cell in ( A ) (*). AnV, Annexin V.

    Techniques Used: Imaging, Knock-Out, Concentration Assay, Fluorescence

    Lack of TRPV4-TMEM16F coupling in BeWo trophoblast cells in the absence of GSK101. ( A ) Outside-out patch recording of BeWo cells in the absence of GSK101. (1) 0 extracellular Ca 2+ ; (2) 2.5 mM extracellular Ca 2+ . 0.2 mM EGTA was included in the pipette. ( B ) Comparison of outside-out patch current with ( Figure 5B , #2, with 2.5 mM Ca 2+ ) and without GSK101 after 1 s of pre-pulse. ( C ) Comparison of outside-out patch current with ( Figure 5B , #1, with 0 Ca 2+ ) and without GSK101 after 1 s of pre-pulse. Values represent mean ± SEM and statistics were done using Student’s t-test (n=5, ****: p
    Figure Legend Snippet: Lack of TRPV4-TMEM16F coupling in BeWo trophoblast cells in the absence of GSK101. ( A ) Outside-out patch recording of BeWo cells in the absence of GSK101. (1) 0 extracellular Ca 2+ ; (2) 2.5 mM extracellular Ca 2+ . 0.2 mM EGTA was included in the pipette. ( B ) Comparison of outside-out patch current with ( Figure 5B , #2, with 2.5 mM Ca 2+ ) and without GSK101 after 1 s of pre-pulse. ( C ) Comparison of outside-out patch current with ( Figure 5B , #1, with 0 Ca 2+ ) and without GSK101 after 1 s of pre-pulse. Values represent mean ± SEM and statistics were done using Student’s t-test (n=5, ****: p

    Techniques Used: Transferring

    TRPV4 and TMEM16F are spatially close to each other on BeWo cell membrane.
    Figure Legend Snippet: TRPV4 and TMEM16F are spatially close to each other on BeWo cell membrane.

    Techniques Used:

    4α-phorbol-12, 13-didecanoate (4α-PDD, 20 μM), a TRPV4 agonist, triggers intracellular Ca 2+ elevation in BeWo cells. ( A ) Ca 2+ dye (Calbryte 520, green) was used to monitor the dynamics of intracellular Ca 2+ . All fluorescence images are the representatives of at least three biological replicates. ( B ) Time course of 4α-PDD triggered Ca 2+ influx in BeWo cells (n=7).
    Figure Legend Snippet: 4α-phorbol-12, 13-didecanoate (4α-PDD, 20 μM), a TRPV4 agonist, triggers intracellular Ca 2+ elevation in BeWo cells. ( A ) Ca 2+ dye (Calbryte 520, green) was used to monitor the dynamics of intracellular Ca 2+ . All fluorescence images are the representatives of at least three biological replicates. ( B ) Time course of 4α-PDD triggered Ca 2+ influx in BeWo cells (n=7).

    Techniques Used: Fluorescence

    TMEM16F CaPLSase and channel activity in BeWo cells is not affected by TRPV4 knockdown. ( A ) Representative images showing 5 μM ionomycin triggers similar levels of PS exposure in scrambled, TPRV4 siRNA2, and siRNA4 knockdown BeWo cells. ( B ) Quantification of ionomycin-induced AnV intensities increase in BeWo cells transfected with scrambled siRNA (n=25), TRPV4 siRNA2 (n=25), or siRNA4 (n=27). All fluorescence images are the representatives of at least three biological replicates. ( C ) Representative TMEM16F current traces from control siRNA and the TRPV4 siRNAs treated BeWo cells recorded with whole-cell patch clamp or patch clamp-lipid scrambling fluorometry (PCLSF). 1000 μM Ca 2+ was included in the pipette solution, and the current was elicited by a voltage step protocol from –100 to +160 mV with holding potential at –60 mV. ( D ) I-V relation of TMEM16F current recorded from the control siRNA and the TRPV4 siRNAs treated BeWo cells. ( E ) Statistics of the TMEM16F current density at +160 mV shown in ( D ). Student’s t-test (ns: not significant, n=7 for control siRNA, n=8 for TRPV4 siRNA2, and n=7 for TRPV4 siRNA4). ( F ) Representative lipid scrambling activities recorded using PCLSF to simultaneously record TMEM16F channel and lipid scramblase activities. The cells are the same ones shown in ( C ). n=3. AnV, Annexin V.
    Figure Legend Snippet: TMEM16F CaPLSase and channel activity in BeWo cells is not affected by TRPV4 knockdown. ( A ) Representative images showing 5 μM ionomycin triggers similar levels of PS exposure in scrambled, TPRV4 siRNA2, and siRNA4 knockdown BeWo cells. ( B ) Quantification of ionomycin-induced AnV intensities increase in BeWo cells transfected with scrambled siRNA (n=25), TRPV4 siRNA2 (n=25), or siRNA4 (n=27). All fluorescence images are the representatives of at least three biological replicates. ( C ) Representative TMEM16F current traces from control siRNA and the TRPV4 siRNAs treated BeWo cells recorded with whole-cell patch clamp or patch clamp-lipid scrambling fluorometry (PCLSF). 1000 μM Ca 2+ was included in the pipette solution, and the current was elicited by a voltage step protocol from –100 to +160 mV with holding potential at –60 mV. ( D ) I-V relation of TMEM16F current recorded from the control siRNA and the TRPV4 siRNAs treated BeWo cells. ( E ) Statistics of the TMEM16F current density at +160 mV shown in ( D ). Student’s t-test (ns: not significant, n=7 for control siRNA, n=8 for TRPV4 siRNA2, and n=7 for TRPV4 siRNA4). ( F ) Representative lipid scrambling activities recorded using PCLSF to simultaneously record TMEM16F channel and lipid scramblase activities. The cells are the same ones shown in ( C ). n=3. AnV, Annexin V.

    Techniques Used: Activity Assay, Transfection, Fluorescence, Patch Clamp, Transferring

    15) Product Images from "Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells"

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20143378

    TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p
    Figure Legend Snippet: TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p

    Techniques Used: Concentration Assay

    TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p
    Figure Legend Snippet: TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p

    Techniques Used:

    TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.
    Figure Legend Snippet: TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.

    Techniques Used: Derivative Assay, Expressing, Knock-Out, Mouse Assay, Fluorescence

    TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p
    Figure Legend Snippet: TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p

    Techniques Used: Derivative Assay, Expressing, Purification, Staining, Concentration Assay, Positive Control

    LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p
    Figure Legend Snippet: LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p

    Techniques Used: Immunofluorescence, Microscopy, Staining

    16) Product Images from "Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion, Phosphatidylinositol-( Phosphatidylinositol-("

    Article Title: Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion, Phosphatidylinositol-( Phosphatidylinositol-(

    Journal: bioRxiv

    doi: 10.1101/2021.12.11.472241

    Ca 2+ influx through TRPV4 activates TMEM16F scramblase. (A) 20 nM GSK101 triggered Ca 2+ influx and PS exposure in BeWo cells. (B) 20 nM GSK101 induced Ca 2+ influx but failed to trigger PS exposure in the TMEM16F knockout (KO) BeWo cell line. Ca 2+ dye (Calbryte 520, green) and fluorescently tagged AnV proteins (AnV-CF594, red) were used to monitor the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( C-D ) Time course of GSK101 triggered Ca 2+ influx (C) and PS exposure (D) in BeWo WT and TMEM16F KO cells.
    Figure Legend Snippet: Ca 2+ influx through TRPV4 activates TMEM16F scramblase. (A) 20 nM GSK101 triggered Ca 2+ influx and PS exposure in BeWo cells. (B) 20 nM GSK101 induced Ca 2+ influx but failed to trigger PS exposure in the TMEM16F knockout (KO) BeWo cell line. Ca 2+ dye (Calbryte 520, green) and fluorescently tagged AnV proteins (AnV-CF594, red) were used to monitor the dynamics of intracellular Ca 2+ and PS externalization, respectively. All fluorescence images are the representatives of at least three biological replicates. ( C-D ) Time course of GSK101 triggered Ca 2+ influx (C) and PS exposure (D) in BeWo WT and TMEM16F KO cells.

    Techniques Used: Knock-Out, Fluorescence

    TRPV4 and TMEM16F are functionally coupled within microdomain (A) Outside-out patch configuration and voltage protocols used to demonstrate TRPV4-TMEM16F coupling. The holding potential was set at −60 mV. A −100 mV pre-pulse with varied length from 0.1 second to 1 second was applied along with perfusion of 30 nM GSK101 to induce Ca 2+ influx. Following the pre-pulse, a depolarized pulse with 0.2 second duration and 140 mV amplitude was applied to record TMEM16F current. (B) Representative outside-out patch recordings from wildtype (WT) BeWo cells under four different conditions: 1) intracellular 0.2 mM EGTA, extracellular 0 Ca 2+ + 30 nM GSK101 (n=5); 2) intracellular 0.2 mM EGTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=5); 3) intracellular 2 mM EGTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=6); 4) intracellular 2 mM BAPTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=6). (C) Diagrams to demonstrate TRPV4-TMEM16F coupling under each condition in Panel B. The intensity of green color depicts Ca 2+ chelating capacity and kinetics.
    Figure Legend Snippet: TRPV4 and TMEM16F are functionally coupled within microdomain (A) Outside-out patch configuration and voltage protocols used to demonstrate TRPV4-TMEM16F coupling. The holding potential was set at −60 mV. A −100 mV pre-pulse with varied length from 0.1 second to 1 second was applied along with perfusion of 30 nM GSK101 to induce Ca 2+ influx. Following the pre-pulse, a depolarized pulse with 0.2 second duration and 140 mV amplitude was applied to record TMEM16F current. (B) Representative outside-out patch recordings from wildtype (WT) BeWo cells under four different conditions: 1) intracellular 0.2 mM EGTA, extracellular 0 Ca 2+ + 30 nM GSK101 (n=5); 2) intracellular 0.2 mM EGTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=5); 3) intracellular 2 mM EGTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=6); 4) intracellular 2 mM BAPTA, extracellular 2.5 mM Ca 2+ + 30 nM GSK101 (n=6). (C) Diagrams to demonstrate TRPV4-TMEM16F coupling under each condition in Panel B. The intensity of green color depicts Ca 2+ chelating capacity and kinetics.

    Techniques Used:

    The activation of TRPV4 elicits TMEM16F current. (A-B) Time course of whole-cell currents elicited in response to 30 nM GSK101 in WT and TMEM16F-KO BeWo cells. The currents were elicited with a ramp protocol shown in Panel B (top). (A) The current amplitudes at +100 mV were plotted every 5 seconds. (B) Representative currents at three different time points as shown in Panel A. (C) Representative current traces elicited by a voltage step protocol (200 ms) from −100 mV to +140 mV at three different time points t1, t2 and t3 as indicated in Panel A. (D) Statistical analysis of current density at t3 in WT and TMEM16F-KO BeWo cells. Values represent mean ± SEM and statistics were done using student t-test (n=5 for each group, **: p
    Figure Legend Snippet: The activation of TRPV4 elicits TMEM16F current. (A-B) Time course of whole-cell currents elicited in response to 30 nM GSK101 in WT and TMEM16F-KO BeWo cells. The currents were elicited with a ramp protocol shown in Panel B (top). (A) The current amplitudes at +100 mV were plotted every 5 seconds. (B) Representative currents at three different time points as shown in Panel A. (C) Representative current traces elicited by a voltage step protocol (200 ms) from −100 mV to +140 mV at three different time points t1, t2 and t3 as indicated in Panel A. (D) Statistical analysis of current density at t3 in WT and TMEM16F-KO BeWo cells. Values represent mean ± SEM and statistics were done using student t-test (n=5 for each group, **: p

    Techniques Used: Activation Assay

    siRNA knockdown of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation. (A) CaPLSase activity was diminished in TRPV4 knockdown BeWo cells. In BeWo cells with TRPV4 knockdown, fluorescently-tagged AnV, an indicator for externalized PS, and Ca 2+ dye show no intensity change after the stimulation of GSK101. (B) CaPLSase activity and intracellular Ca 2+ increasing induced by 20 nM GSK101 was eliminated by 500 nM GSK219. (C) Time course of whole-cell currents elicited in response to GSK101 in scramble siRNA (control) or TRPV4 siRNAs treated BeWo cells. The currents were elicited with a ramp protocol from −100 mV to +100 mV and plotted every 5 seconds at +100 mV. (D) Representative current traces elicited by a voltage step protocol (200 ms) from −100 mV to +140 mV at three different time points t1, t2 and t3 as indicated on panel C. (E) Statistical analysis of current density at t3 in WT and TRPV4-siRNA knockdown BeWo cells. Current densities after scrambled siRNA, TRPV4-siRNA2 and TRPV4-siRNA4 treated are 84.72±21.97, 11.12±2.03 and 7.32±2.53 pA/pF, respectively. Values represent mean ± SEM and statistics were done using student t-test (n=5 for each group, ***: p
    Figure Legend Snippet: siRNA knockdown of TRPV4 abolishes GSK101-induced Ca 2+ influx and subsequent TMEM16F activation. (A) CaPLSase activity was diminished in TRPV4 knockdown BeWo cells. In BeWo cells with TRPV4 knockdown, fluorescently-tagged AnV, an indicator for externalized PS, and Ca 2+ dye show no intensity change after the stimulation of GSK101. (B) CaPLSase activity and intracellular Ca 2+ increasing induced by 20 nM GSK101 was eliminated by 500 nM GSK219. (C) Time course of whole-cell currents elicited in response to GSK101 in scramble siRNA (control) or TRPV4 siRNAs treated BeWo cells. The currents were elicited with a ramp protocol from −100 mV to +100 mV and plotted every 5 seconds at +100 mV. (D) Representative current traces elicited by a voltage step protocol (200 ms) from −100 mV to +140 mV at three different time points t1, t2 and t3 as indicated on panel C. (E) Statistical analysis of current density at t3 in WT and TRPV4-siRNA knockdown BeWo cells. Current densities after scrambled siRNA, TRPV4-siRNA2 and TRPV4-siRNA4 treated are 84.72±21.97, 11.12±2.03 and 7.32±2.53 pA/pF, respectively. Values represent mean ± SEM and statistics were done using student t-test (n=5 for each group, ***: p

    Techniques Used: Activation Assay, Activity Assay

    TRPV4 is functionally expressed in human trophoblasts. ( A ) Schematic of the first trimester placental villus and trophoblast fusion. (B-C) qRT-PCR of TRPV4 in primary human trophoblasts (B) and BeWo cells (C). All genes were normalized to GAPDH and then normalized to Syncytin-1 (SYN1). (D) Representative immunofluorescence of TRPV4 (green) and nuclei (blue) in a human first trimester placenta villus (cross-section). TRPV4 is expressed in both cytotrophoblasts and syncytiotrophoblasts. (E) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue). All fluorescence images are the representatives of at least three biological replicates. (F) GSK1016790A (GSK101, 20 nM), a specific TRPV4 agonist, triggered robust intracellular Ca 2+ elevation in BeWo cells. (G) GSK2193874 (GSK219, 500 nM), a selective TRPV4 antagonist, completely prevented GSK101-induced Ca 2+ influx through TRPV4 channels in BeWo cells. Ca 2+ elevation through ionomycin was intact in the presence of GSK219. (H) Summary of GSK101 and GSK219 effects on BeWo cell Ca 2+ dynamics measured by Ca 2+ dye (Calbryte 520). All fluorescence images are the representatives of at least three biological replicates. (I-J) Time course of outside-out currents elicited in response to 30 nM GSK101 with and without 500 nM GSK219 (I) and representative current traces in Panel I in the presence of GSK101 and GSK219+GSK101 (J). The currents were elicited by a 500 ms ramp voltage protocol from −100 mV to +100 mV. The holding potential was set at −60 mV.
    Figure Legend Snippet: TRPV4 is functionally expressed in human trophoblasts. ( A ) Schematic of the first trimester placental villus and trophoblast fusion. (B-C) qRT-PCR of TRPV4 in primary human trophoblasts (B) and BeWo cells (C). All genes were normalized to GAPDH and then normalized to Syncytin-1 (SYN1). (D) Representative immunofluorescence of TRPV4 (green) and nuclei (blue) in a human first trimester placenta villus (cross-section). TRPV4 is expressed in both cytotrophoblasts and syncytiotrophoblasts. (E) Representative immunofluorescence of TRPV4 (green) in BeWo cells. The nuclei were stained with Hoechst (blue). All fluorescence images are the representatives of at least three biological replicates. (F) GSK1016790A (GSK101, 20 nM), a specific TRPV4 agonist, triggered robust intracellular Ca 2+ elevation in BeWo cells. (G) GSK2193874 (GSK219, 500 nM), a selective TRPV4 antagonist, completely prevented GSK101-induced Ca 2+ influx through TRPV4 channels in BeWo cells. Ca 2+ elevation through ionomycin was intact in the presence of GSK219. (H) Summary of GSK101 and GSK219 effects on BeWo cell Ca 2+ dynamics measured by Ca 2+ dye (Calbryte 520). All fluorescence images are the representatives of at least three biological replicates. (I-J) Time course of outside-out currents elicited in response to 30 nM GSK101 with and without 500 nM GSK219 (I) and representative current traces in Panel I in the presence of GSK101 and GSK219+GSK101 (J). The currents were elicited by a 500 ms ramp voltage protocol from −100 mV to +100 mV. The holding potential was set at −60 mV.

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence

    TRPV4 inhibition hinders trophoblast fusion in vitro but not in vivo . (A) Representative images of control and GSK219 treated BeWo cells after 48-hour forskolin treatment. Nuclei and membranes were labelled with Hoechst (blue) and Di-8-ANEPPS (green), respectively. (B) GSK219 inhibits forskolin-induced BeWo cell fusion. Unpaired two-sided Student’s t -test. ***: p
    Figure Legend Snippet: TRPV4 inhibition hinders trophoblast fusion in vitro but not in vivo . (A) Representative images of control and GSK219 treated BeWo cells after 48-hour forskolin treatment. Nuclei and membranes were labelled with Hoechst (blue) and Di-8-ANEPPS (green), respectively. (B) GSK219 inhibits forskolin-induced BeWo cell fusion. Unpaired two-sided Student’s t -test. ***: p

    Techniques Used: Inhibition, In Vitro, In Vivo

    17) Product Images from "TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway"

    Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-021-02315-8

    TRPV4 activation led to enhanced expression of TNF receptor 1 (TNFR1). A Immunoblotting analysis showing that pre-injection of GSK101 enhanced the expression of TNFR1 in the retina, compared with the control group. B Bar chart summarizing mean expression levels of TNFR1 under different conditions. n = 5, p
    Figure Legend Snippet: TRPV4 activation led to enhanced expression of TNF receptor 1 (TNFR1). A Immunoblotting analysis showing that pre-injection of GSK101 enhanced the expression of TNFR1 in the retina, compared with the control group. B Bar chart summarizing mean expression levels of TNFR1 under different conditions. n = 5, p

    Techniques Used: Activation Assay, Expressing, Injection

    TRPV4 activation led to increased phosphorylation of JAK2 and STAT3, thereby inducing NF-κB p65 translocation from the cytoplasm into the nucleus. A , C Immunoblotting analysis showing that GSK101 treatment increased the phosphorylation of STAT3 and JAK2, compared with the control group, but had no effect on the protein levels of JAK2 and STAT3 ( C ). B , D Bar charts summarizing mean expression levels of phosphorylated STAT3/STAT ( B ) and phosphorylated JAK2/JAK2 ( D ) under different conditions. n = 3 for all groups, * p
    Figure Legend Snippet: TRPV4 activation led to increased phosphorylation of JAK2 and STAT3, thereby inducing NF-κB p65 translocation from the cytoplasm into the nucleus. A , C Immunoblotting analysis showing that GSK101 treatment increased the phosphorylation of STAT3 and JAK2, compared with the control group, but had no effect on the protein levels of JAK2 and STAT3 ( C ). B , D Bar charts summarizing mean expression levels of phosphorylated STAT3/STAT ( B ) and phosphorylated JAK2/JAK2 ( D ) under different conditions. n = 3 for all groups, * p

    Techniques Used: Activation Assay, Translocation Assay, Expressing

    Müller cells express functional TRPV4. A Immunofluorescence images show that cells with red autofluorescence are Müller cells. A1 Immunofluorescence images show cells with red autofluorescence in rat retinal vertical slices acquired from TdTomato transgenic mouse retina (Tomato). A2 Immunofluorescence images show glutamine synthetase (GS) protein staining (green) in the slices depicted in A1 . A3 Immunofluorescence images show DAPI (blue) staining in the slices depicted in A1 . A4 shows merged images. Scale bar: 50 µm. B Representative trace recorded from a Müller cell identified by spontaneous red fluorescence, showing that perfusion with GSK101 (10 μM) caused significant depolarization of Müller membrane potential. C Bar chart showing GSK101-induced depolarization of Müller cell membrane potential. n = 4. * p
    Figure Legend Snippet: Müller cells express functional TRPV4. A Immunofluorescence images show that cells with red autofluorescence are Müller cells. A1 Immunofluorescence images show cells with red autofluorescence in rat retinal vertical slices acquired from TdTomato transgenic mouse retina (Tomato). A2 Immunofluorescence images show glutamine synthetase (GS) protein staining (green) in the slices depicted in A1 . A3 Immunofluorescence images show DAPI (blue) staining in the slices depicted in A1 . A4 shows merged images. Scale bar: 50 µm. B Representative trace recorded from a Müller cell identified by spontaneous red fluorescence, showing that perfusion with GSK101 (10 μM) caused significant depolarization of Müller membrane potential. C Bar chart showing GSK101-induced depolarization of Müller cell membrane potential. n = 4. * p

    Techniques Used: Functional Assay, Immunofluorescence, Transgenic Assay, Staining, Fluorescence

    TRPV4 activation enhances TNF-α production in retinal tissues. A Cumulative changes in TNF-α mRNA levels in saline-injected retinas (control) and retinas with GSK101 injection at 1 week. n = 5, * p
    Figure Legend Snippet: TRPV4 activation enhances TNF-α production in retinal tissues. A Cumulative changes in TNF-α mRNA levels in saline-injected retinas (control) and retinas with GSK101 injection at 1 week. n = 5, * p

    Techniques Used: Activation Assay, Injection

    TRPV4 activation enhances the expression of GFAP. A Immunofluorescence images show GFAP protein expression profiles in rat retinal vertical slices acquired from sham-operated retinas (saline-injected; control) ( A1 ), 1 µM GSK101-injected retinas ( A2 ), and 10 µM GSK101-injected retinas ( A3 ). Retinas that received no GFAP antibody served as negative controls ( A4 ). Double immunofluorescence staining showing GFAP expression when the GFAP antibody was pre-adsorbed with its blocking peptide (BP) ( A5 ). Scale bar: 20 µm. B Representative immunoblots showing changes in GFAP protein levels in control and 10 µM GSK101-injected retinas. C Bar chart summarizing mean expression levels of GFAP under different conditions. n = 6. * p
    Figure Legend Snippet: TRPV4 activation enhances the expression of GFAP. A Immunofluorescence images show GFAP protein expression profiles in rat retinal vertical slices acquired from sham-operated retinas (saline-injected; control) ( A1 ), 1 µM GSK101-injected retinas ( A2 ), and 10 µM GSK101-injected retinas ( A3 ). Retinas that received no GFAP antibody served as negative controls ( A4 ). Double immunofluorescence staining showing GFAP expression when the GFAP antibody was pre-adsorbed with its blocking peptide (BP) ( A5 ). Scale bar: 20 µm. B Representative immunoblots showing changes in GFAP protein levels in control and 10 µM GSK101-injected retinas. C Bar chart summarizing mean expression levels of GFAP under different conditions. n = 6. * p

    Techniques Used: Activation Assay, Expressing, Immunofluorescence, Injection, Double Immunofluorescence Staining, Blocking Assay, Western Blot

    TNF-α inhibition reduces TRPV4-mediated retinal cell apoptosis. A1 – A2 DAPI staining in GSK101-injected ( A1 ) and R7050 with GSK101-injected (R7050 + GSK101) ( A2 ) whole flat-mounted retinas at 7 days after injections in the regions at angle 0°. A3 – A4 Counterstained images with TUNEL staining detection of cell apoptosis (green). A5 – A6 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 µm (for all images). B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. R7050 (1 µM, 2 μl) was pre-injected 1 day before the GSK101 injection. n = 5. ** p
    Figure Legend Snippet: TNF-α inhibition reduces TRPV4-mediated retinal cell apoptosis. A1 – A2 DAPI staining in GSK101-injected ( A1 ) and R7050 with GSK101-injected (R7050 + GSK101) ( A2 ) whole flat-mounted retinas at 7 days after injections in the regions at angle 0°. A3 – A4 Counterstained images with TUNEL staining detection of cell apoptosis (green). A5 – A6 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 µm (for all images). B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. R7050 (1 µM, 2 μl) was pre-injected 1 day before the GSK101 injection. n = 5. ** p

    Techniques Used: Inhibition, Staining, Injection, TUNEL Assay

    TRPV4 activation enhances TNF-α production in cultured Müller cells. A Morphology of cultured Müller cells. Scale bar: 50 µm. B GSK101 treatment enhanced GFAP protein levels in cultured Müller cells. n = 4. ** p
    Figure Legend Snippet: TRPV4 activation enhances TNF-α production in cultured Müller cells. A Morphology of cultured Müller cells. Scale bar: 50 µm. B GSK101 treatment enhanced GFAP protein levels in cultured Müller cells. n = 4. ** p

    Techniques Used: Activation Assay, Cell Culture

    Changes in TRPV4 protein levels in retinas of rats with COH. A Representative immunoblots showing changes in TRPV4 protein levels in control and COH retinal extracts at different postoperative times (G1w, G2w, and G3w). B Bar chart summarizing mean expression levels of TRPV4 at different postoperative times. Data are presented as means ± standard errors of the mean. n = 7, 4, 6, and 5, respectively. ** p
    Figure Legend Snippet: Changes in TRPV4 protein levels in retinas of rats with COH. A Representative immunoblots showing changes in TRPV4 protein levels in control and COH retinal extracts at different postoperative times (G1w, G2w, and G3w). B Bar chart summarizing mean expression levels of TRPV4 at different postoperative times. Data are presented as means ± standard errors of the mean. n = 7, 4, 6, and 5, respectively. ** p

    Techniques Used: Western Blot, Expressing

    Schematic diagram showing the signaling pathway involved in TRPV4 activation-mediated TNF-α production in Müller cells and RGC apoptosis in COH retinas. NF-κB nuclear factor-kappa B, TNF-α tumor necrosis factor-α, TNFR1 TNF receptor 1
    Figure Legend Snippet: Schematic diagram showing the signaling pathway involved in TRPV4 activation-mediated TNF-α production in Müller cells and RGC apoptosis in COH retinas. NF-κB nuclear factor-kappa B, TNF-α tumor necrosis factor-α, TNFR1 TNF receptor 1

    Techniques Used: Activation Assay

    Effects of GSK101 on retinal ganglion cell (RGC) apoptosis and survival. A TRPV4 activation leads to enhanced RGC apoptosis. A1 – A3 4′,6-diamidino-2-phenylindole (DAPI) staining in saline-injected (control) ( A1 ), GSK101-injected (GSK101) ( A2 ), and HC-067 with GSK101 (HC) ( A3 ) retinas at 1 week after injection in the regions at angle 0°. Images were acquired from whole flat-mounted retinal preparations. A4 – A6 , Counterstained images with TUNEL staining detection of RGC apoptosis (green). A7 – A9 , Merged images of corresponding TUNEL and DAPI images. Scale bar: 50 µm. B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 5 for all groups. C Pre-application of HC-067 reduces RGC apoptosis in COH retinas. C1 – C3 DAPI staining in sham-operated (control) ( C1 ), COH ( C2 ), and HC-067 with COH (HC + COH) ( C3 ) retinas at G2w. C4 – C6 Counterstained images with TUNEL staining detection of RGC apoptosis (green). C7 – C9 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 μm. D Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 6 for all groups. * p
    Figure Legend Snippet: Effects of GSK101 on retinal ganglion cell (RGC) apoptosis and survival. A TRPV4 activation leads to enhanced RGC apoptosis. A1 – A3 4′,6-diamidino-2-phenylindole (DAPI) staining in saline-injected (control) ( A1 ), GSK101-injected (GSK101) ( A2 ), and HC-067 with GSK101 (HC) ( A3 ) retinas at 1 week after injection in the regions at angle 0°. Images were acquired from whole flat-mounted retinal preparations. A4 – A6 , Counterstained images with TUNEL staining detection of RGC apoptosis (green). A7 – A9 , Merged images of corresponding TUNEL and DAPI images. Scale bar: 50 µm. B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 5 for all groups. C Pre-application of HC-067 reduces RGC apoptosis in COH retinas. C1 – C3 DAPI staining in sham-operated (control) ( C1 ), COH ( C2 ), and HC-067 with COH (HC + COH) ( C3 ) retinas at G2w. C4 – C6 Counterstained images with TUNEL staining detection of RGC apoptosis (green). C7 – C9 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 μm. D Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 6 for all groups. * p

    Techniques Used: Activation Assay, Staining, Injection, TUNEL Assay

    18) Product Images from "Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat"

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2012.e32

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.
    Figure Legend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Techniques Used: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.
    Figure Legend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Techniques Used: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P
    Figure Legend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Techniques Used: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation

    19) Product Images from "Piezo1-mediated stellate cell activation causes pressure-induced pancreatic fibrosis in mice"

    Article Title: Piezo1-mediated stellate cell activation causes pressure-induced pancreatic fibrosis in mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.158288

    TRPV4-KO mice were protected from pancreatic duct ligation–induced fibrosis. ( A and D ) DIC and Bodipy 493/503–stained images of PSCs from TRPV4-KO mice 24 hours after Yoda1 (25 μM). ( B and C ) Mean cell area and Feret’s diameter (max) of PSCs 24 hours after Yoda1 (25 μM) (from 3 experiments with 20 cells each). ( E ) Loss of fat droplets in PSCs following Yoda1 (25 μM) (from 3 experiments and > 100 cells). ( F and G ) Quantification of collagen type I and fibronectin immunostaining in PSCs from TRPV4-KO mice 4 days after Yoda1 (25 μM). ( H and I ) Representative images of collagen type I and fibronectin staining for the data shown in F and G . ( J – M ) Pancreatic duct ligation (PDL) at the tail region of the pancreas induced chronic pancreatitis and fibrosis in WT and TRPV4-KO mice. Eight days after PDL, chronic pancreatitis and fibrosis parameters of the tail region included ( J ) H E staining, ( K ) H E score, ( L ) Masson’s trichrome staining, and ( M ) area of WT and TRPV4-KO mice ( n = 5). Statistical comparisons were made using 2-tailed Student’s t test. *** P ≤ 0.001. Scale bar: 100 μm.
    Figure Legend Snippet: TRPV4-KO mice were protected from pancreatic duct ligation–induced fibrosis. ( A and D ) DIC and Bodipy 493/503–stained images of PSCs from TRPV4-KO mice 24 hours after Yoda1 (25 μM). ( B and C ) Mean cell area and Feret’s diameter (max) of PSCs 24 hours after Yoda1 (25 μM) (from 3 experiments with 20 cells each). ( E ) Loss of fat droplets in PSCs following Yoda1 (25 μM) (from 3 experiments and > 100 cells). ( F and G ) Quantification of collagen type I and fibronectin immunostaining in PSCs from TRPV4-KO mice 4 days after Yoda1 (25 μM). ( H and I ) Representative images of collagen type I and fibronectin staining for the data shown in F and G . ( J – M ) Pancreatic duct ligation (PDL) at the tail region of the pancreas induced chronic pancreatitis and fibrosis in WT and TRPV4-KO mice. Eight days after PDL, chronic pancreatitis and fibrosis parameters of the tail region included ( J ) H E staining, ( K ) H E score, ( L ) Masson’s trichrome staining, and ( M ) area of WT and TRPV4-KO mice ( n = 5). Statistical comparisons were made using 2-tailed Student’s t test. *** P ≤ 0.001. Scale bar: 100 μm.

    Techniques Used: Mouse Assay, Ligation, Staining, Immunostaining

    Piezo1 mediates TRPV4 channel opening in pancreatic stellate cells. ( A ) Immunostaining of GFAP and TRPV4 in human PSCs. ( B ) TRPV4 agonist GSK101 (100 nM) effects on [Ca 2+ ] i in human PSCs with and without the TRPV4 blocker HC067 (1 μM). ( C ) GSK101 (100 nM) effects on [Ca 2+ ] i in PSCs from 3 biological samples were blocked with the TRPV4 antagonist HC067 (1 μM) (from 18 to 37 cells). ( D ) Immunostaining of GFAP and TRPV4 in mouse PSCs. ( E and F ) Traces and graph represent the effects of the TRPV4 agonist GSK101 (100 nM) on [Ca 2+ ] i in mouse PSCs with and without the TRPV4 blocker HC067 (1 μM) (from 18 cells). ( G – I ) Effects of Yoda1 (25 μM) on [Ca 2+ ] i in PSCs from WT and TRPV4-KO mice. ( I ) Statistical analyses of peak and sustained [Ca 2+ ] i elevation (from 24 to 32 cells). The sustained [Ca 2+ ] i elevation was measured at 6 minutes after Yoda1. ( J and K ) Effects of phospholipase A2 blockers AACOCF3 (30 μM) and YM26734 (10 μM) on Yoda1-induced (25 μM) [Ca 2+ ] i in PSCs (from 24 to 26 cells). The sustained calcium rise was measured at 8 minutes after Yoda1 application. ( L – N ) Fluid shear stress (12 dyne/cm 2 ) was applied for 1 minute in PSCs from WT and TRPV4-KO mice and TRPV4-KO mice with GsMTx4 (5 μM). In panels G , H , and L – N , each colored line represents the response of a single cell. ( O ) Quantification of peak [Ca 2+ ] i following shear stress (12 dyne/cm 2 ) for 1 minute in TRPV4-KO PSCs with and without GsMTx4 (from 24 cells). Statistical analyses were calculated using 2-tailed Student’s t test. * P ≤ 0.05 and **** P ≤ 0.0001. Scale bar: 10 μm.
    Figure Legend Snippet: Piezo1 mediates TRPV4 channel opening in pancreatic stellate cells. ( A ) Immunostaining of GFAP and TRPV4 in human PSCs. ( B ) TRPV4 agonist GSK101 (100 nM) effects on [Ca 2+ ] i in human PSCs with and without the TRPV4 blocker HC067 (1 μM). ( C ) GSK101 (100 nM) effects on [Ca 2+ ] i in PSCs from 3 biological samples were blocked with the TRPV4 antagonist HC067 (1 μM) (from 18 to 37 cells). ( D ) Immunostaining of GFAP and TRPV4 in mouse PSCs. ( E and F ) Traces and graph represent the effects of the TRPV4 agonist GSK101 (100 nM) on [Ca 2+ ] i in mouse PSCs with and without the TRPV4 blocker HC067 (1 μM) (from 18 cells). ( G – I ) Effects of Yoda1 (25 μM) on [Ca 2+ ] i in PSCs from WT and TRPV4-KO mice. ( I ) Statistical analyses of peak and sustained [Ca 2+ ] i elevation (from 24 to 32 cells). The sustained [Ca 2+ ] i elevation was measured at 6 minutes after Yoda1. ( J and K ) Effects of phospholipase A2 blockers AACOCF3 (30 μM) and YM26734 (10 μM) on Yoda1-induced (25 μM) [Ca 2+ ] i in PSCs (from 24 to 26 cells). The sustained calcium rise was measured at 8 minutes after Yoda1 application. ( L – N ) Fluid shear stress (12 dyne/cm 2 ) was applied for 1 minute in PSCs from WT and TRPV4-KO mice and TRPV4-KO mice with GsMTx4 (5 μM). In panels G , H , and L – N , each colored line represents the response of a single cell. ( O ) Quantification of peak [Ca 2+ ] i following shear stress (12 dyne/cm 2 ) for 1 minute in TRPV4-KO PSCs with and without GsMTx4 (from 24 cells). Statistical analyses were calculated using 2-tailed Student’s t test. * P ≤ 0.05 and **** P ≤ 0.0001. Scale bar: 10 μm.

    Techniques Used: Immunostaining, Mouse Assay

    20) Product Images from "TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats"

    Article Title: TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats

    Journal: JCI Insight

    doi: 10.1172/jci.insight.137528

    Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P
    Figure Legend Snippet: Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats. ( A ) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.( B ) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. ( C ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. ( D ) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( A – D ); * P

    Techniques Used: Expressing, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P
    Figure Legend Snippet: TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3. ( A ) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. ( B ) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( D ); * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, BrdU Staining

    The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P
    Figure Legend Snippet: The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats. ( A ) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. ( B ) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. ( C and D ) Representative time course ( C ) of the changes in [Ca 2+ ] i and quantification ( D ) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test ( B ) and 1-way ANOVA with Bonferroni’s post hoc test ( D ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats. ( A ) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. ( B and C ) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B and C ); ** P

    Techniques Used:

    Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P
    Figure Legend Snippet: Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts. ( A – C ) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy ( B ), fibrillation ( C ), and none of both ( A ) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. ( C ) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. ( D ) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ 2 test; * P

    Techniques Used: Isolation, Activation Assay

    Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P
    Figure Legend Snippet: Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats. ( A ) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. ( B – D ) Mean rest membrane potential (RMP) and AP amplitude (APA) ( B ), AP slope ( C ), and action potential duration (APD) ( D ) until 20%, 50%, and 90% of repolarization (APD 20 , APD 50 , and APD 70 , respectively) in atrial myocytes. ( E ) Voltage clamp protocol. ( F ) Representative the outward voltage-gated K + currents ( I K ) recorded from isolated atrial myocytes of indicated groups.( G – I ) Mean current-voltage (I–V) curves for the peak ( I peak , G ), sustained ( I ss , H ), and transient ( I to , I ). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B , C , D , G , H , and I ); * P

    Techniques Used: Isolation

    Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P
    Figure Legend Snippet: Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ); * P

    Techniques Used: Western Blot

    Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P
    Figure Legend Snippet: Upregulated expression of TRPV4 in the atria of SP rats and AF patients. ( A and B ) Representative Western blot ( A ) and quantification ( B ) of TRPV4 in atrial tissue of sham ( n = 14) and SP rats 1 day (d) ( n = 5), 2 d ( n = 7), 3 d ( n = 6), 4 d ( n = 6), 5 d ( n = 6), 7 d ( n = 6), and 14 d ( n = 5) after surgery. ( C ) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. ( D and E ) Representative Western blot ( D ) and quantification ( E ) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF ( n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test ( B ) and 2-tailed unpaired Student’s t test ( E ); * P

    Techniques Used: Expressing, Western Blot, Immunohistochemistry, Negative Control

    21) Product Images from "Electroacupuncture at Hua Tuo Jia Ji Acupoints Reduced Neuropathic Pain and Increased GABAA Receptors in Rat Spinal Cord"

    Article Title: Electroacupuncture at Hua Tuo Jia Ji Acupoints Reduced Neuropathic Pain and Increased GABAA Receptors in Rat Spinal Cord

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/8041820

    Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat SC. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in the SC of the rats from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p
    Figure Legend Snippet: Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat SC. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in the SC of the rats from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p

    Techniques Used: Expressing

    Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat SSC. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in the SSC of rats from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p
    Figure Legend Snippet: Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat SSC. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in the SSC of rats from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p

    Techniques Used: Expressing

    Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat DRG. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in DRG from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p
    Figure Legend Snippet: Expression levels of GABA A , A1R, TRPV1, TRPV4, and mGluR3 receptors in rat DRG. (a) GABA A , (b) A1R, (c) TRPV1, (d) TRPV4, and (e) mGluR3 expression levels in DRG from the sham, 2-Hz EA, and 15-Hz EA groups (from left to right). Sham: neuropathic pain rats with sham EA; 2 Hz: neuropathic pain rats that received 2-Hz EA; 15 Hz: neuropathic pain rats that received 15-Hz EA. ∗p

    Techniques Used: Expressing

    22) Product Images from "Determinants of TRPV4 Activity following Selective Activation by Small Molecule Agonist GSK1016790A"

    Article Title: Determinants of TRPV4 Activity following Selective Activation by Small Molecule Agonist GSK1016790A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016713

    Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells. A–C: HeLa-TRPV4 cells in Ca 2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B . Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C . Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E : HeLa-TRPV4 cells in Ca 2+ free media (Na + containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca 2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca 2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca 2+ -containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca 2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.
    Figure Legend Snippet: Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells. A–C: HeLa-TRPV4 cells in Ca 2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B . Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C . Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E : HeLa-TRPV4 cells in Ca 2+ free media (Na + containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca 2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca 2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca 2+ -containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca 2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.

    Techniques Used: Patch Clamp, Activation Assay

    Effect of GSK101 on the plasma membrane expression of TRPV4 channel in HeLa-TRPV4 cells, over the time course of 30 min. A. Western blots showing that GSK101 caused a downregulation of TRPV4 in the pure plasma membrane fractions, while having no significant effect on the TRPV4 abundance in the post-nuclear (whole cell) lysates. B. Densitometry of the normalized TRPV4/cadherin ratio on the plasma membrane showed the statistically significant downregulation of TRPV4 under GSK101 treatment in HeLa-TRPV4 cells (Mean ± SE, n = 5, P
    Figure Legend Snippet: Effect of GSK101 on the plasma membrane expression of TRPV4 channel in HeLa-TRPV4 cells, over the time course of 30 min. A. Western blots showing that GSK101 caused a downregulation of TRPV4 in the pure plasma membrane fractions, while having no significant effect on the TRPV4 abundance in the post-nuclear (whole cell) lysates. B. Densitometry of the normalized TRPV4/cadherin ratio on the plasma membrane showed the statistically significant downregulation of TRPV4 under GSK101 treatment in HeLa-TRPV4 cells (Mean ± SE, n = 5, P

    Techniques Used: Expressing, Western Blot

    Dose-dependent activation of Ca 2+ influx under GSK101 stimulation. HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca 2+ ] i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC 50 = 3.3 nM.
    Figure Legend Snippet: Dose-dependent activation of Ca 2+ influx under GSK101 stimulation. HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca 2+ ] i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC 50 = 3.3 nM.

    Techniques Used: Activation Assay, Imaging

    FRET analysis of TRPV4 channel under GSK101 (10 nM) treatment. HeLa cells were co-transfected with TRPV4-mCerulean (donor, in blue) and TRPV4-mVenus (acceptor, in yellow), and fixed at 0, 3, 10 and 30 min after GSK101 treatment. A . Pre-bleach and post-bleach images of TRPV4-mCerulean and TRPV4-mVenus overlapping with each other. Post-bleach image lost TRPV4-mVenus (from laser bleaching at 514 nm) and appeared bluer due to the increase of energy in TRPV4-mCerulean. The red box PN represented the perinuclearROI, the white box M represented the plasma membrane ROI. B. Summary of FRET efficiency between TRPV4-mCerulean and TRPV4-mVenus under three independent experiment sets of GSK101 stimulation. There was no significant different in FRET efficiency among the time point groups, both in PN and M regions (Mean ± SE, n = 3, P > 0.05). However, FRET efficiency was significantly higher in the M region than in the PN region, in 0 and 10 min post GSK treatment groups (Mean ± SE, n = 3, P
    Figure Legend Snippet: FRET analysis of TRPV4 channel under GSK101 (10 nM) treatment. HeLa cells were co-transfected with TRPV4-mCerulean (donor, in blue) and TRPV4-mVenus (acceptor, in yellow), and fixed at 0, 3, 10 and 30 min after GSK101 treatment. A . Pre-bleach and post-bleach images of TRPV4-mCerulean and TRPV4-mVenus overlapping with each other. Post-bleach image lost TRPV4-mVenus (from laser bleaching at 514 nm) and appeared bluer due to the increase of energy in TRPV4-mCerulean. The red box PN represented the perinuclearROI, the white box M represented the plasma membrane ROI. B. Summary of FRET efficiency between TRPV4-mCerulean and TRPV4-mVenus under three independent experiment sets of GSK101 stimulation. There was no significant different in FRET efficiency among the time point groups, both in PN and M regions (Mean ± SE, n = 3, P > 0.05). However, FRET efficiency was significantly higher in the M region than in the PN region, in 0 and 10 min post GSK treatment groups (Mean ± SE, n = 3, P

    Techniques Used: Transfection

    Effects of ruthenium red (3 µM) and Ca 2+ free media on GSK101-induced intracellular Ca 2+ elevation in HeLa-TRPV4 cells. A. Ruthenium red (RR) at 3 µM partially inhibited GSK101-induced intracellular Ca 2+ elevation when added early (1–3 min post stimulation), while having less inhibitory effect if added at a later time point (10 min post stimulation). Ionomycin (5 µM) added on top of RR caused a significant Ca 2+ influx in these cells. B. Ca 2+ free media abolished GSK101-induced intracellular Ca 2+ elevation when added early (3 min post stimulation), while having a much smaller effect if added at a later time point (10 min post stimulation). Ionomycin (5 µM) added while the cells were in Ca 2+ free media caused a transient increase in intracellular Ca 2+ concentration followed by the trending down of intracellular Ca 2+ . C.Summary bar graph showing the percentage inhibition of RR and Ca 2+ free media added at early or late stage of GSK101 activation, respectively. The inhibition at the late stage displayed a much smaller percentage inhibition for either RR or Ca 2+ free media(Mean ± SE, P
    Figure Legend Snippet: Effects of ruthenium red (3 µM) and Ca 2+ free media on GSK101-induced intracellular Ca 2+ elevation in HeLa-TRPV4 cells. A. Ruthenium red (RR) at 3 µM partially inhibited GSK101-induced intracellular Ca 2+ elevation when added early (1–3 min post stimulation), while having less inhibitory effect if added at a later time point (10 min post stimulation). Ionomycin (5 µM) added on top of RR caused a significant Ca 2+ influx in these cells. B. Ca 2+ free media abolished GSK101-induced intracellular Ca 2+ elevation when added early (3 min post stimulation), while having a much smaller effect if added at a later time point (10 min post stimulation). Ionomycin (5 µM) added while the cells were in Ca 2+ free media caused a transient increase in intracellular Ca 2+ concentration followed by the trending down of intracellular Ca 2+ . C.Summary bar graph showing the percentage inhibition of RR and Ca 2+ free media added at early or late stage of GSK101 activation, respectively. The inhibition at the late stage displayed a much smaller percentage inhibition for either RR or Ca 2+ free media(Mean ± SE, P

    Techniques Used: Concentration Assay, Inhibition, Activation Assay

    Effects of 10 nM GSK101 on intracellular Ca 2+ concentration in HeLa-TRPV4 cells using calcium imaging analysis. A. The representative Ca 2+ trace of HeLa cells (average of 15-20 cells from one experiment) transiently transfected with the empty mVenus-N1 vector (HeLamVenus) showed no response to GSK101 stimulation (negative control). The representative Ca 2+ trace of HeLa TRPV4-mVenus cells showed a rapid increase in intracellular Ca 2+ concentration under GSK101 stimulation, reaching its maximum level within 2 minutes and sustained for over 30 min. B.Summary bar graph showing statistically significant difference in delta [Ca 2+ ] i between HeLamVenus cells and HeLa TRPV4-mVenus cells (Mean ± SE, n = 3–6, P
    Figure Legend Snippet: Effects of 10 nM GSK101 on intracellular Ca 2+ concentration in HeLa-TRPV4 cells using calcium imaging analysis. A. The representative Ca 2+ trace of HeLa cells (average of 15-20 cells from one experiment) transiently transfected with the empty mVenus-N1 vector (HeLamVenus) showed no response to GSK101 stimulation (negative control). The representative Ca 2+ trace of HeLa TRPV4-mVenus cells showed a rapid increase in intracellular Ca 2+ concentration under GSK101 stimulation, reaching its maximum level within 2 minutes and sustained for over 30 min. B.Summary bar graph showing statistically significant difference in delta [Ca 2+ ] i between HeLamVenus cells and HeLa TRPV4-mVenus cells (Mean ± SE, n = 3–6, P

    Techniques Used: Concentration Assay, Imaging, Transfection, Plasmid Preparation, Negative Control

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    Alomone Labs antibody anti trpv4 extracellular
    PAR2 and <t>TRPV4</t> expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.
    Antibody Anti Trpv4 Extracellular, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti rat trpv4
    Confocal microscope images of double-immunofluorescence staining of mesenteric resistant arteries (A, B, C, G, H, I) and DRG neurons (D, E, F). A, D and G: FITC-labeled <t>TRPV4</t> receptor staining (green). B and E: Cy3-labeled CGRP staining (red). C and F: colocalization of TRPV4 and CGRP (yellow). H: Cy3-labeled MaxiК channels staining (red). I: colocalization of TRPV4 and MaxiК channels (yellow). Blue arrows: the colocalized areas. Negative control not shown. Scale bars, 20 μm.
    Rabbit Anti Rat Trpv4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs trpv4
    Microscopic images showing localization of <t>TRPV4</t> in duck sperm. (A-B) Confocal microscopic images depicting the localization of TRPV4 (green) as detected by two different antibodies and Nucleus (blue) by DAPI. Mitochondria (red) is labelled by Mitotracker Red dye in A and C to highlight the channel expression in the mitochondrial region. ( C ) SR-SIM images of TRPV4 localization (using Ab1 antibody) at the head (left) and tail (right) of duck sperm is shown. ( D ) Zoomed up image of neck region of sperm depicting the presence of TRPV4 (green) in the neck region. The head (blue) and arrows mark the start and end point of mitochondrial region.
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    PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Expressing, Immunohistochemistry

    PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Activation Assay

    TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Activity Assay

    Confocal microscope images of double-immunofluorescence staining of mesenteric resistant arteries (A, B, C, G, H, I) and DRG neurons (D, E, F). A, D and G: FITC-labeled TRPV4 receptor staining (green). B and E: Cy3-labeled CGRP staining (red). C and F: colocalization of TRPV4 and CGRP (yellow). H: Cy3-labeled MaxiК channels staining (red). I: colocalization of TRPV4 and MaxiК channels (yellow). Blue arrows: the colocalized areas. Negative control not shown. Scale bars, 20 μm.

    Journal: Journal of hypertension

    Article Title: Hypotension Induced by TRPV4 Activation: Role of Ca2+-activated K+ Channels and Sensory Nerves

    doi: 10.1097/HJH.0b013e328332b865

    Figure Lengend Snippet: Confocal microscope images of double-immunofluorescence staining of mesenteric resistant arteries (A, B, C, G, H, I) and DRG neurons (D, E, F). A, D and G: FITC-labeled TRPV4 receptor staining (green). B and E: Cy3-labeled CGRP staining (red). C and F: colocalization of TRPV4 and CGRP (yellow). H: Cy3-labeled MaxiК channels staining (red). I: colocalization of TRPV4 and MaxiК channels (yellow). Blue arrows: the colocalized areas. Negative control not shown. Scale bars, 20 μm.

    Article Snippet: After washing with PBS, the arteries were incubated at 4 °C overnight in a primary antibody cocktail of goat anti-rat MaxiКβ (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-rat TRPV4 (1:100, Alomone labs, Jerusalem, Israel), followed by a secondary antibody cocktail of donkey anti-goat CY3 (1:136, Jackson ImmunoResearch labs, West Grove, PA) and donkey anti-rabbit FITC (1:136, Jackson ImmunoResearch labs, West Grove, PA) for 30 minutes.

    Techniques: Microscopy, Double Immunofluorescence Staining, Labeling, Staining, Negative Control

    Microscopic images showing localization of TRPV4 in duck sperm. (A-B) Confocal microscopic images depicting the localization of TRPV4 (green) as detected by two different antibodies and Nucleus (blue) by DAPI. Mitochondria (red) is labelled by Mitotracker Red dye in A and C to highlight the channel expression in the mitochondrial region. ( C ) SR-SIM images of TRPV4 localization (using Ab1 antibody) at the head (left) and tail (right) of duck sperm is shown. ( D ) Zoomed up image of neck region of sperm depicting the presence of TRPV4 (green) in the neck region. The head (blue) and arrows mark the start and end point of mitochondrial region.

    Journal: bioRxiv

    Article Title: Differential expression and localization of thermosensitive Transient Receptor Potential Vanilloid (TRPV) channels in the mature sperm of white pekin duck (Anas platyrhynchos)

    doi: 10.1101/2020.02.10.941732

    Figure Lengend Snippet: Microscopic images showing localization of TRPV4 in duck sperm. (A-B) Confocal microscopic images depicting the localization of TRPV4 (green) as detected by two different antibodies and Nucleus (blue) by DAPI. Mitochondria (red) is labelled by Mitotracker Red dye in A and C to highlight the channel expression in the mitochondrial region. ( C ) SR-SIM images of TRPV4 localization (using Ab1 antibody) at the head (left) and tail (right) of duck sperm is shown. ( D ) Zoomed up image of neck region of sperm depicting the presence of TRPV4 (green) in the neck region. The head (blue) and arrows mark the start and end point of mitochondrial region.

    Article Snippet: Confocal microscopy using two different antibodies against the C-terminus of TRPV4 we found that TRPV4 is very scarce in the head, prominent at the mitochondrial region (seen by its colocalization with MitoTracker Red, indicated by white arrows) and at the tail of duck sperm ( ).

    Techniques: Expressing

    Prevalence of physiologically relevant thermosensitive TRPV channels in mature duck sperm. Flow cytometric evaluation of duck sperm stained for physiologically relevant thermosensitive TRPV channels are shown. A. Representative dot-plots showing percentage of cells expressing TRPV1, TRPV3, TRPV4 channels detected by Ab-1 (antibodies from Alomone labs) antibody specific for each TRPV channel. B. Histograms showing percentage of cells expressing TRPV channels and corresponding Mean Fluorescence Intensity (MFI) of TRPV channels detected by Ab2 antibody of each channel (from Sigma Aldrich), expressed as fold change in comparison to MFI of unstained cells. n = 3, unpaired T-test. ** = P

    Journal: bioRxiv

    Article Title: Differential expression and localization of thermosensitive Transient Receptor Potential Vanilloid (TRPV) channels in the mature sperm of white pekin duck (Anas platyrhynchos)

    doi: 10.1101/2020.02.10.941732

    Figure Lengend Snippet: Prevalence of physiologically relevant thermosensitive TRPV channels in mature duck sperm. Flow cytometric evaluation of duck sperm stained for physiologically relevant thermosensitive TRPV channels are shown. A. Representative dot-plots showing percentage of cells expressing TRPV1, TRPV3, TRPV4 channels detected by Ab-1 (antibodies from Alomone labs) antibody specific for each TRPV channel. B. Histograms showing percentage of cells expressing TRPV channels and corresponding Mean Fluorescence Intensity (MFI) of TRPV channels detected by Ab2 antibody of each channel (from Sigma Aldrich), expressed as fold change in comparison to MFI of unstained cells. n = 3, unpaired T-test. ** = P

    Article Snippet: Confocal microscopy using two different antibodies against the C-terminus of TRPV4 we found that TRPV4 is very scarce in the head, prominent at the mitochondrial region (seen by its colocalization with MitoTracker Red, indicated by white arrows) and at the tail of duck sperm ( ).

    Techniques: Staining, Expressing, Fluorescence

    Endogenous expression of TRPV channels in duck sperm. Western blot analysis of duck sperm extracts probed with different TRPV-specific antibodies are shown. A. TRPV1 specific band is detected by a specific antibody (directed against the C-terminus of TRPV1, Alomone Labs) in absence but not in presence of its blocking peptide; B. Western blot analysis with antibody that detects TRPV2 (raised against the C-terminus, Alomone Labs). C. Two different antibodies detecting TRPV3 [raised against the C-terminus, (Ab1: Alomone Labs) and N-terminus (Ab3: Sigma Aldrich)] detect similar expression pattern of TRPV3. D. Two different antibodies raised against the TRPV4 [raised against C-terminus, Ab1: Alomone Labs) and N-terminus (Ab3: Sigma Aldrich)] detect TRPV4 at the expected size. E. Two different antibodies raised against the C-terminus of TRPV5 (Ab1: Alomone Labs and Ab2: Sigma-Aldrich) detects TRPV5 at expected size. F. A specific antibody raised against the C-terminus of TRPV6 (Ab-1: Alomone Labs) detects TRPV6 in absence but not in presence of its blocking peptide.

    Journal: bioRxiv

    Article Title: Differential expression and localization of thermosensitive Transient Receptor Potential Vanilloid (TRPV) channels in the mature sperm of white pekin duck (Anas platyrhynchos)

    doi: 10.1101/2020.02.10.941732

    Figure Lengend Snippet: Endogenous expression of TRPV channels in duck sperm. Western blot analysis of duck sperm extracts probed with different TRPV-specific antibodies are shown. A. TRPV1 specific band is detected by a specific antibody (directed against the C-terminus of TRPV1, Alomone Labs) in absence but not in presence of its blocking peptide; B. Western blot analysis with antibody that detects TRPV2 (raised against the C-terminus, Alomone Labs). C. Two different antibodies detecting TRPV3 [raised against the C-terminus, (Ab1: Alomone Labs) and N-terminus (Ab3: Sigma Aldrich)] detect similar expression pattern of TRPV3. D. Two different antibodies raised against the TRPV4 [raised against C-terminus, Ab1: Alomone Labs) and N-terminus (Ab3: Sigma Aldrich)] detect TRPV4 at the expected size. E. Two different antibodies raised against the C-terminus of TRPV5 (Ab1: Alomone Labs and Ab2: Sigma-Aldrich) detects TRPV5 at expected size. F. A specific antibody raised against the C-terminus of TRPV6 (Ab-1: Alomone Labs) detects TRPV6 in absence but not in presence of its blocking peptide.

    Article Snippet: Confocal microscopy using two different antibodies against the C-terminus of TRPV4 we found that TRPV4 is very scarce in the head, prominent at the mitochondrial region (seen by its colocalization with MitoTracker Red, indicated by white arrows) and at the tail of duck sperm ( ).

    Techniques: Expressing, Western Blot, Blocking Assay

    Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    doi: 10.4081/ejh.2012.e32

    Figure Lengend Snippet: Effects of hypotonicity on the distribution of TRPV4 in ventricular myocytes. Iso and Hypo: isotonic and hypotonic bath solutions, respectively. A, B) Immuno-localization of TRPV4 protein in cultured ventricular myocytes before (A) and after (B) hypotonic stimulation (scale bar: 25 µm). The myocytes were doubly labeled for TRPV4 protein (A-1, B-1) and the nucleus (A-2, B-2) as did as in Figure 1 . A-3 and B-3 were correspondingly overlaid images. C, D) Immunoreactivity of TRPV4 protein detected by immuno-electron microscopy in cultured ventricular myocytes before (C) and after (D) hypotonic stimulation. N, nucleus; C, cytoplasm; arrows indicate the colloidal gold granules.

    Article Snippet: It also seems unlikely that a different protein with the same molecular weight could bind with the anti-TRPV4 antibody.

    Techniques: Cell Culture, Labeling, Immuno-Electron Microscopy

    Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    doi: 10.4081/ejh.2012.e32

    Figure Lengend Snippet: Localization of TRPV4 protein in cardiac myocytes. A, B) Confocal images of freshly isolated (A1–3, scale bar: 15 µm) and cultured neonatal ventricular myocytes (B1–3, scale bar: 25 µm) labeled with anti-TRPV4 antibody (A1 and B1) and DAPI (A2 and B2), respectively, and the merged images (A3 and B3). The freshly isolated neonatal ventricular myocyte was small and round. C) Confocal images of freshly isolated adult rat ventricular myocytes labeled with anti-TRPV4 antibody (scale bar: 25 µm). D) Confocal image of the cultured ventricular myocytes in blank control (without TRPV4 antibody). E) Confocal image of the cultured ventricular myocytes in absorption test, in which the anti-TRPV4 antibody was preincubated with the peptide antigen. F, G) Immunolabeling TRPV4 protein in sections of the neonatal and adult ventricles.

    Article Snippet: It also seems unlikely that a different protein with the same molecular weight could bind with the anti-TRPV4 antibody.

    Techniques: Isolation, Cell Culture, Labeling, Immunolabeling

    Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Journal: European Journal of Histochemistry : EJH

    Article Title: Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

    doi: 10.4081/ejh.2012.e32

    Figure Lengend Snippet: Hypotonically induced translocation of TRPV4 protein in cultured ventricular myocytes. A) The TRPV4 mRNA transcript was detected in adult renal tissues and cultured neonatal ventricular myocytes by RT-PCR amplification. B) Quantification of TRPV4 mRNA by real-time PCR for cultured ventricular myocytes in isotonic bath solution (Iso) and after hypotonic stimulation (Hypo). There were no significant differences at the mRNA levels between the two groups. C) Western blot analysis on the total TRPV4 protein of the freshly isolated adult ventricular myocytes and the corresponding absorption test. D) Western blot analysis on the total TRPV4 protein of cultured neonatal ventricular myocytes before and after exposure to hypotonic stimulation. E) Western blot analysis on TRPV4 protein in the nucleus fraction before and after hypotonic stimulation. F) Total and nuclear TRPV4 protein under isotonic and hypotonic conditions. The longitudinal coordinate stands for the relative ratio of TRPV4 fluorescent value contrast to β-actin fluorescent value (*P

    Article Snippet: It also seems unlikely that a different protein with the same molecular weight could bind with the anti-TRPV4 antibody.

    Techniques: Translocation Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Isolation