Journal: Life

Article Title: The Effect of Citral on Aquaporin 5 and Trpv4 Expressions and Uterine Contraction in Rat—An Alternative Mechanism

doi: 10.3390/life11090897

Figure Lengend Snippet: Changes in AQP5 mRNA ( A ) and protein ( B , 35 kDa) and TRPV4 mRNA ( C ) and protein ( D , 105 kDa) expression of on gestational day 22 in pregnant rat uteri after citral treatment, in vitro. β-actin (43 kDa) was used as a loading control. C : control uterus, Cit : citral-treated uterus, ns > 0.05; *** p < 0.001; compared to the control uterus sample. n = 6.

Article Snippet: The following primers were used: assay ID Rn00562837_m1 for the Aqp5 water channel, Rn00576745_m1 for Trpv4 and Rn00667869_m1 for β-actin as endogenous control (Thermo Fisher Scientific, Hungary).

Techniques: Expressing, In Vitro, Control

Journal: Cellular oncology (Dordrecht, Netherlands)

Article Title: TRPV4 drives the progression of leiomyosarcoma by promoting ECM1 generation and co-activating the FAK/PI3K/AKT/GSK3β pathway.

doi: 10.1007/s13402-024-01008-7

Figure Lengend Snippet: Fig. 6 TRPV4 induces LMS progres sion via FAK/PI3K/AKT/GSK3β signaling. A Schematic workflow for transcriptomic analysis and proteomic analysis of SK-UT-1 cells infected with TRPV4-OE or Vector. B The top ten up-regulated KEGG pathway related to the genes in SK-UT-1 cells transfected with TRPV4-OE or Vector. C Venn dia gram of transcriptomic analysis and proteomic analysis in SK-UT-1 cells with up-regulated genes. D Protein expression of TRPV4, ECM1, FAK, pFAK, PI3K, pPI3K, AKT, pAKT, GSK3β, pGSK-3β, β-catenin and GAPDH in LMS cells. SK-UT-1 cells were infected with TRPV4-OE or Vector, MES-SA with shTRPV4-1 or NC. E Protein expression of ECM1, FAK, pFAK, PI3K, pPI3K, AKT, pAKT, GSK3β, pGSK-3β, β-catenin and GAPDH in LMS cells. The cells were transfected with ECM1-OE or VEC. F Protein expression of PI3K, pPI3K, AKT, pAKT, GSK3β, pGSK-3β, β-catenin and GAPDH in LMS cells. The TRPV4 overexpres sion tumor cells were untreated or treated with LY (20 µM). G Repre sentative data from Transwell migra tion and invasion assays performed in the indicated cells treated with or without LY (scale bar, 200 μm). H and I ECM1 protein levels in the supernatant of LMS cells. J Relative expression levels of FAK, pFAK, PI3K, pPI3K, AKT, pAKT, GSK3β, pGSK-3β, β-catenin and GAPDH in the indicated cells treated with or without recombinant ECM1 protein. *indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001. Data are mean ± SD or representatives; n = 3 independent experiments

Article Snippet: The diluted primary antibodies were as follows: GAPDH (1: 100000, Proteintech, Hubei, China), TRPV4 (1:1000, Abcam, Cambridge, UK), ECM1 (1: 1000, Proteintech), AKT (1:1000, Cell Signal Technology, Boston, MA, USA), pAKT (1:1000, Cell Signal Technology), FAK (1:1000, Cell Signal Technology), pFAK (1:1000, Cell Signal Technology), PI3K (1:1000, Proteintech), pPI3K (1:1000, Abmart, Shanghai, China), GSK3β (1:1000, Cell Signal Technology), pGSK3β (1:1000, Cell Signal Technology), β-catenin (1:1000, Abcam).

Techniques: Infection, Plasmid Preparation, Transfection, Expressing, Recombinant

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Role of TRPV4 and K + channels in AA‐induced dilation of HCAs. A, AA dilated HCAs in a concentration‐dependent manner. The dilation was markedly inhibited by the TRPV4 antagonist RN‐1734 (20 μmol/L). B, AA‐induced dilation was inhibited by the combination of TRAM‐34 (1 μmol/L) and apamin (1 μmol/L), which selectively inhibit intermediate‐conductance K Ca (IK Ca ) and small‐conductance K Ca (SK Ca ) channels, respectively. C, AA‐induced dilation was eliminated by high extracellular K + (80 mmol/L); n=5 to 7 patients/each group. * P <0.05 vs control. TRPV4 indicates transient receptor potential vanilloid 4; AA, arachidonic acid; HCA, human coronary artery.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Concentration Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: AA‐induced increase of [Ca 2+ ] i in native HCAECs. A, AA (3 μmol/L) increased [Ca 2+ ] i , which was nearly eliminated in the absence of extracellular Ca 2+ (Ca 2+ free). B, AA‐elicited [Ca 2+ ] i increase was inhibited by 3 CYP pathway inhibitors: ETYA (30 μmol/L), 17‐ODYA (10 μmol/L), and MS‐PPOH (30 μmol/L). C, Treatment of cells with TRPV4 antagonists RN‐1734 (20 μmol/L), HC‐067047 (1 μmol/L), and ruthenium red (RuR; 1 μmol/L) also markedly inhibited AA‐induced [Ca 2+ ] i elevation. D, The TRPV4 agonist GSK1016790A (GSK; 10 nmol/L) increased [Ca 2+ ] i , which was inhibited by RN‐1734. All data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05, ** P <0.01 versus control. AA indicates arachidonic acid; ETYA, eicosatetraynoic acid; 17‐ODYA, 17‐octadecynoic acid; MS‐PPOH, N‐(methylsulfonyl)‐2‐(2‐propynyloxy)‐benzenehexanamide; HCAEC, human coronary artery endothelial cell; CYP, cytochrome P450; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Ca 2+ responses in hTRPV4‐overexpressing HCAECs. A, Representative images of endothelial cells expressing hTRPV4‐GFP (top) and corresponding cells loaded with fura‐2 before (middle) and after (bottom) AA treatment. B, Compared with nontransduced (NT) cells, AA (3 μmol/L) elicited augmented [Ca 2+ ] i elevation in hTRPV4‐expressing cells. C, The AA‐induced Ca 2+ response was inhibited by CYP pathway inhibitors ETYA (30 μmol/L) and 17‐ODYA (10 μmol/L). D, None of 4 isomeric EETs (3 or 10 μmol/L in some experiments) showed substantial activation of TRPV4 channels compared with AA. All data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05 versus control. hTRPV4 indicates human transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; GFP, green fluorescent protein; AA, arachidonic acid; CYP, cytochrome P450; EET, epoxyeicosatrienoic acid.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Expressing, Activation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Modulation of TRPV4‐mediated Ca 2+ influx by membrane potential. A, AA (3 μmol/L)‐elicited [Ca 2+ ] i increase was blunted in the presence of 60 mmol/L extracellular K + . B, AA induced membrane hyperpolarization in HCAECs, as indicated by a decrease in PMPI fluorescence intensity (F480). In contrast, the TRPV4 agonist 4α‐PDD (3 μmol/L) depolarized the membrane potential. Valinomycin (K + ‐selective ionophore, 2 μmol/L) was used as a positive control to indicate membrane hyperpolarization. C, The TRPV4 specific agonist 4α‐PDD (5 μmol/L) increased [Ca 2+ ] i in HCAECs, and the increase was not affected by 60 mmol/L K + . D, Membrane hyperpolarization by valinomycin resulted in Ca 2+ influx in hTRPV4‐expressing HCAECs, which was inhibited by HC‐067047. The data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05 vs control, ** P <0.01 vs vehicle, † P <0.05 vs valinomycin (5 μmol/L). TRPV4 indicates transient receptor potential vanilloid 4; AA, arachidonic acid; 4α‐PDD, 4α‐phorbol‐12,13‐didecanoate; HCAEC, human coronary artery endothelial cell; PMPI, plasma membrane potential indicator.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Fluorescence, Positive Control, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Regulation of TRPV4 activation by protein phosphorylation. A, In hTRPV4‐expressing HCAECs, the AA‐induced [Ca 2+ ] i increase was almost abolished by the PKA inhibitor PKI (1 μmol/L), whereas this AA response was only partially but not significantly attenuated by the PKC inhibitor GF 109203X (1 μmol/L). B, TRPV4 is phosphorylated at serine‐824 in unstimulated HCAECs expressing the hTRPV4‐GFP fusion protein. The level of serine‐824 phosphorylation was not further increased by AA (3 μmol/L). PKI (1 μmol/L) significantly inhibited serine‐824 phosphorylation. Endothelial cells were treated with the indicated reagents, and TRPV4‐GFP was immunoprecipitated with GFP antibodies and detected with pS824 antibodies (upper panel). The same membrane was reprobed with GFP antibodies to detect total TRPV4 protein (middle panel). All data represent mean±SEM from 3 to 5 independent experiments. At least 60 cells analyzed in (A). * P <0.05, ** P <0.01 vs control. TRPV4 indicates transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; AA, arachidonic acid; PKA, protein kinase A; PKI, protein kinase A inhibitor; IP, immunoprecipitation; IB, immunoblotting; GFP, green fluorescent protein.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Activation Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Single‐channel properties of hTRPV4 overexpressed in HCAECs. A, TRPV4 single‐channel currents (left) were recorded from a cell‐attached patch at the indicated membrane potentials (Vm). Right panel depicts average current–voltage relationship for single‐channel currents, with a calculated slope conductance determined over voltage ranges of 0 to 60 and −40 to 0 mV, respectively. Data are mean±SE from 5 membrane patches. B, Single‐channel activity of TRPV4 (left) was low under basal conditions but markedly increased following bath perfusion of the TRPV4 agonist 4α‐PDD (1 μmol/L). Shown on the right are the corresponding amplitude histograms in relation to open‐state probability (NPo). Data are representative of >5 membrane patches. Dashed lines indicate current level when the channel is closed. Cell‐attached recordings were obtained with a normal extracellular solution (140 Na + , 5 Cs + ) in the pipette and a high‐K + (140 K + ) bath solution to zero the cell membrane potential. TRPV4 indicates transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Activity Assay, Transferring

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Effect of AA on hTRPV4 single‐channel currents in HCAECs. AA (1 μmol/L) increased TRPV4 single‐channel currents in both cell‐attached patches (A) and inside‐out patches, which lacked intracellular constituents (B). Left, representative recordings at the indicated membrane potential (Vm). Note that c is the current level when the channel is closed. Right, summary of channel open‐state probability (NPo). Cell‐attached and inside‐out patch recordings before and after treatment with AA were performed with a normal extracellular solution (140 Na + ,5 Cs + ) in the pipette and a high‐K + (140 K + ) solution in the bath. n=6 patches/each group. * P <0.05 vs control. C, Possible mechanism for AA‐induced Ca 2+ entry through TRPV4 channels in human coronary endothelial cells and subsequent dilation of coronary arterioles. AA indicates arachidonic acid; TRPV4, transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; PKA, protein kinase A; EDH, endothelium‐dependent hyperpolarization.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Transferring

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Phylogeny of the TRPV4 in vertebrates and expression of different Trpv4 splice variants in seabream testis and spermatozoa. a Bayesian majority rule consensus tree (1 million MCMC generations, aamodel = mixed) of a ClustalX amino acid alignment of Trpv4 protein sequences rooted with the ghost shark Trpv4. The GenBank accession numbers of the sequences are listed in Supplementary Table . Bayesian posterior probabilities are shown at each node with the scale bar indicating expected substitution rates per site. b Schematic representation of the gilthead seabream trpv4 exons, and the polypeptides of wild-type Trpv4 (Trpv4_v1) and splice variants Trpv4_v2 and _v10 (Ensembl accession numbers ENSSAUT00010061485.1, ENSSAUT00010061488.1 and ENSSAUT00010061519.1, respectively). The proline-rich domain (PRD) and ankyrin repeat domain (ARD) are shown for each protein. c Representative RT-PCR detection of seabream trpv4_v1, _v2 and _v10 mRNAs in testis and ejaculated spermatozoa (SPZ). The N line is the negative control (absence of RT during cDNA synthesis). The arrows indicate the specific amplified transcripts, and the sizes (kb) of molecular markers are indicated on the left. The positions on the trpv4 genomic sequence of the different oligonucleotide primers employed are indicated in b

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Both Trpv4 and Aqp4a are expressed in seabream spermatozoa. a Immunostaining (left panels, bright field [BF]; right panels, epifluorescence images) of Trpv4 in ejaculated spermatozoa using two commercial rabbit antibodies against mammalian TRPV4 (α-TRPV4-1 [Novus Biologicals # NBP2-41262] and α-TRPV4-2 [Invitrogen # OSR00136W]), showing that the Trpv4 immunoreaction (red color) was localized along the flagellum (arrows). The nucleus is counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. b Immunoblots of total membrane protein extracts from X. laevis oocytes uninjected (Uninj.), oocytes injected with cRNA encoding seabream Trpv4_v1, and sperm flagella (Sp), using the two TRPV4 antibodies. Spermatozoa extracts were treated with or without PNGase F (plus or minus) prior to electrophoresis. Arrows indicate monomers, while the asterisks indicate glycosylated forms. The arrowheads indicate other potential post-translational modifications of the ion channel. Molecular mass markers (kDa) are on the left. c Immunolocalization of seabream Aqp4a (green) along the tail of the spermatozoa (arrows) using a paralog-specific chicken antibody (α-Aqp4a). The negative controls (right panels) were incubated with the primary antibody preadsorbed with the antigenic peptide. Labels and scale bars as in A. d Schematic diagram of the Aqp4a topology depicting the cytoplasmatic N- and C-termini (NT and CT, respectively), the six transmembrane α-helices (1–6), and the five loops ( A-E ). The two translation initiating methionines (M1 and M43) in the NT are shown. e, f Immunoblot of X. laevis oocytes uninjected (Uninj) or expressing the Aqp4a-M1 or Aqp4a-M43 isoforms ( e ), and of sperm flagella showing the expression of both isoforms (arrows) ( f ). For the latter immunoblot, the α-Aqp4a was preadsorbed with the antigenic peptide before immunoblotting to verify the specificity of the reactive bands. Labels as in b . g Double immunostaining of Trpv4 and Aqp4a in seabream spermatozoa using the TRPV4-1 and Aqp4a antibodies showing co-localization of both channels (arrows). Labels and scale bars as in a . h Co-immunoprecipitation of Trpv4 and Aqp4a in ejaculated spermatozoa using either of the two commercial TRPV4 antibodies or immunoglobulin G (IgG) as control. The immunoprecipitates were immunoblotted with the TRPV4 or Aqp4a antibodies as indicated. Labels as in b . IgG-HC, IgG heavy chain

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Immunostaining, Western Blot, Membrane, Injection, Electrophoresis, Incubation, Expressing, Double Immunostaining, Immunoprecipitation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Functional characterization of seabream Trpv4 variants in X. laevis oocytes. a Representative double immunostaining of uninjected oocytes and oocytes expressing Aqp4a-M43 together with human influenza hemagglutinin (HA)-tagged Trpv4_v1, _v2 or _v10 using a seabream Aqp4a-specific antiserum and anti-HA antibodies. The oocyte plasma membrane was counterstained with wheat germ agglutinin (WGA). Scale bars, 10 μm. b-c Representative volume ( b ) and current ( c ) traces obtained from oocytes voltage-clamped at Vm = − 20 mV and challenged with a hypo- or hyperosmotic gradient (− 100 mOsm: blue bars and + 100 mOsm: red bars, respectively). The traces were recorded with a 200-ms step protocol (as indicated by numbers 1–4) from an uninjected oocyte and oocytes expressing either Aqp4a, Trpv4_v1, _v2 or _v10 alone, or Aqp4a together with Trpv4_v1, _v2 or _v10. d-f Summarized I/V curves from oocytes expressing Aqp4a plus Tpv4_v1, _v2 or _v10 in control solution (white) or during application of a hyposmotic (blue) or hyperosmotic solution (red). g-i Trpv4_v1, _v2 or _v10-mediated current activity at -85 mV obtained after exposure to -100 mOsm (blue) or + 100 mOsm (red) normalized to that obtained in control conditions. In d-i panels, values ( n = 5–9 oocytes; white dots in g-i ) are presented as mean ± SEM. The paired normalized data in g-i were statistically analyzed by one sample t -test (***, p < 0.001, with respect to the same oocytes at 220 mOsm prior to osmotic challenge; ns, not statistically significant). The controls (uninjected oocytes and oocytes expressing Aqp4a or Tpv4_v1, Trpv4_v2 or Trpv4_v10 alone) and exposed to an isosmotic solution or during application of a hyposmotic or hyperosmotic solution are shown in Supplementary Fig.

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Functional Assay, Double Immunostaining, Expressing, Membrane, Control, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Inhibition of Trpv4_v1 and _v10 with different TRPV4 blockers in X. laevis oocytes. a-l Summarized I/V curves from oocytes expressing either Tpv4_v1 ( a-f ) or _v10 ( g-l ) alone in control solution (white dots) or after 5 min exposure to different TRPV4 blockers at two concentrations, RN-1734 (10 and 100 µM, blue and red dots, respectively), ruthenium red (RR, 1 and 10 µM, blue and red dots, respectively) and HC-067047 (1 and 10 µM, blue and red dots, respectively). m-r The Trpv4_v1- ( m-o ) or _v10- ( p-r ) mediated current activity at -85 mV obtained after exposure to the different TRPV4 blockers was normalized to that obtained in control conditions. In all panels, data are presented as mean ± SEM ( n = 5–6 oocytes; white dots in m-r ). The paired normalized values were statistically analyzed by one sample t -test (*, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to the same oocytes before treatment with the inhibitors; ns, not statistically significant)

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Inhibition, Expressing, Control, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Inhibition of Trpv4 or Aqp4a impairs seabream sperm motility. a-c Inhibition of the percentage of motility and progressivity (% MOT and % PROG, respectively) and curvilinear velocity (VCL) at 5, 30 and 60 s post-activation induced by increasing doses of the TRPV4 antagonist RN-1734 or the Aqp4a antibody (α-Aqp4a), or by 20 µM of TGN-020. Control spermatozoa were treated with 0.5% DMSO (vehicle, a and c ) or 200 nM IgY ( b ). In all panels, the data are the mean ± SEM ( n = 5–8 males, one ejaculate per male; white dots). Statistical differences in a and b within each time point were measured by one-way ANOVA, or Kruskal-Wallis test, followed by Dunn’s multiple comparisons test ( a and b ), or by an unpaired Student t -test ( c ). *, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to non-treated sperm. The brightfield and immunostaining images in panel b confirm the specific binding of α-Aqp4a to Aqp4a in the spermatozoon flagellum through the labelling of either IgY- or α-Aqp4a in vitro-treated spermatozoa with anti-chicken secondary antibodies. The nucleus of the spermatozoa was counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Inhibition, Activation Assay, Control, Immunostaining, Binding Assay, In Vitro

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

doi: 10.1007/s00018-024-05341-w

Figure Lengend Snippet: Model for the RVI mechanism in post-activated seabream spermatozoa. Upon release into the hyperosmotic seawater a rapid water efflux mediated by Aqp1aa results in cell shrinkage and subsequent [Ca 2+ ] i increase, both triggering flagellar motility. The rapid spermatozoon shrinkage could induce the activation of NKCC1 and Na + , K + , Cl − influx, which would then drive water uptake through Aqp4a, and perhaps also by NKCC1 supported water fluxes. Water influx into the spermatozoon could induce local swelling of the flagellum, triggering the activation of the Trpv4 and a further local Ca 2+ influx, which may activate signaling events for the stimulation of L-type Ca 2+ channels, and perhaps of other Ca 2+ channels present in the spermatozoa, thereby allowing a massive Ca 2+ influx. The increased accumulation of Ca 2+ and other ions would facilitate further Aqp4a-mediated water uptake in the spermatozoon, and feasibly also through Aqp1ab1 and/or -10bb present in the anterior region of the flagellum, to promote a fast volume recovery and to maintain motility

Article Snippet: Antibodies against human or mouse TRPV4 were purchased from Novus Biologicals (# NBP2-41262) and Invitrogen (# OSR00136W), respectively.

Techniques: Activation Assay

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Detection of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues by IHC staining of tissue microarrays. ( A ) Representative images of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues. (a, b) Weakly focal positive TRPV4 expression in adjacent noncancerous gastric epithelia. (c, d) Strongly positive TRPV4 expression in a case of gastric adenocarcinoma. ( B ) Scatterplots of IHC scores of TRPV4 expression in gastric adenocarcinoma tissues and adjacent noncancerous tissues. ( C ) Percents of tissue samples with high TRPV4 expression and low TRPV4 expression based on the cut-off value of IHC scores in gastric adenocarcinoma tissues and adjacent noncancerous tissues (***P<0.001). Magnification: (a,c) ×50 and (b,d) ×400. Abbreviations: IHC, immunohistochemical; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Association of TRPV4 Expression with Clinicopathological Characteristics in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Representation images of TRPV4, E-cadherin and vimentin expression in gastric cancer tissues by immunohistochemical staining. Magnification, ×400. ( A – C ) High TRPV4 expression in a case of poor differentiated gastric adenocarcinoma with loss expression of E-cadherin and positive vimentin expression. ( D – F ) Low TRPV4 expression in a case of signet ring cell carcinoma with high E-cadherin expression and negative Vimentin expression. Abbreviation: TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Correlation Between Expression of TRPV4, E-Cadherin and Vimentin in Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Survival curves using the Kaplan–Meier method by Log rank test. ( A and B ) The patients with high TRPV4 expression had shorter overall survival and disease-free survival than those with low TRPV4 expression. ( C and D ) The patients with low E-cadherin expression had shorter overall survival and disease-free survival than those with high E-cadherin expression. ( E and F ) The patients with high vimentin expression had shorter overall survival and disease-free survival than those with low vimentin expression. ( G ) Overall survival analysis of TRPV4 gene in gastric cancer obtained from Kaplan–Meier Plotter online ( http://kmplot.com/analysis/ ). *P<0.05, **P<0.01, ***P<0.001. Abbreviations: Cum, cumulative; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analyses of Overall Survival in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analyses of Disease-Free Survival in Patients with Gastric Cancer

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: The protein expression of TRPV4 in gastric cell lines and down-regulation of TPRV4 inhibited proliferation of gastric cancer cells. ( A ) The protein expression levels of TPRV4 were detected in gastric cancer cell lines (HGC-27 and MGC-803) and normal gastric mucosa cell GES-1 by Western blot. **P<0.01, ***P<0.001. ( B ) Western blot analyses of the expression levels of TRPV4 protein in gastric cancer cell lines following transfection with shTRPV4 or shCtrl. ***P<0.001. ( C ) Proliferation capacities of gastric cancer cells lines were assayed at 24 h, 48 h and 72 h by CCK-8 following transfection with shTRPV4 or shCtrl. ***P<0.001. ( D ) Colony formation assays were performed in gastric cancer cell lines following transfection with shTRPV4 or shCtrl. *P<0.05. Abbreviations: CCK-8, Cell Counting Kit-8; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Western Blot, Transfection, CCK-8 Assay, Cell Counting

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Transwell assays demonstrated that TRPV4 down-regulation greatly inhibited invasion abilities of gastric cancer cells. ( A and B ) Representative images of invasive cells following transfection with shTRPV4 or shCtrl. Magnification, ×200. ( C and D ) The column diagram of the numbers of invasive cells following transfection with shTRPV4 or shCtrl. **P<0.01, ***P<0.001. Abbreviation: TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Transfection

Journal: OncoTargets and therapy

Article Title: TRPV4 Overexpression Promotes Metastasis Through Epithelial–Mesenchymal Transition in Gastric Cancer and Correlates with Poor Prognosis

doi: 10.2147/OTT.S256918

Figure Lengend Snippet: Down-regulation of TPRV4 regulated the expression levels of EMT-related proteins in gastric cancer cell lines ( A ) The expression levels of EMT-related proteins (E-cadherin, vimentin, N-Cadherin, Snail, Slug and Twist) were detected by Western blot assays following transfection with shTRPV4 or shCtrl. ( B ) The column diagram of the expression levels of EMT-related proteins in HGC-27 following transfection with shTRPV4 or shCtrl. ( C ) The column diagram of the expression levels of EMT-related proteins in MGC-803 following transfection with shTRPV4 or shCtrl. *P<0.05, ***P<0.001. Abbreviations: ns, non-significant; EMT, epithelial–mesenchymal transition; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: After blocking with 20% bovine serum albumin (BSA), these pretreated slides were incubated at 4°C overnight with antibodies against TRPV4 (1:100 dilution, #ACC-034-AO, Alomone labs), E-cadherin (1:50 dilution, clone NCH-38, Dako), and vimentin (1:100 dilution, clone V9, Dako), followed by incubation with the HRP-conjugated secondary antibody (Dako).

Techniques: Expressing, Western Blot, Transfection