Journal: Experimental and Therapeutic Medicine

Article Title: A high salt diet impairs the bladder epithelial barrier and activates the NLRP3 and NF‑κB signaling pathways to induce an overactive bladder in vivo

doi: 10.3892/etm.2024.12651

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative PCR.

Article Snippet: The samples were then incubated at 4˚C overnight with the following primary antibodies: Rabbit anti-TJP-1 antibody (1:250; cat. no. ab276131; Abcam), rabbit anti-CLAUDIN-1 antibody (1:200; cat. no. YT0942; ImmunoWay Biotechnology Company), rabbit anti-TRPV4 antibody (1:200; cat. no. YT5833; ImmunoWay Biotechnology Company), rabbit anti-CASP1 antibody (1:200; cat. no. YP0749; ImmunoWay Biotechnology Company) and rabbit anti-NF-κB antibody (1:200; cat. no. YM8001; ImmunoWay Biotechnology Company).

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: A high salt diet impairs the bladder epithelial barrier and activates the NLRP3 and NF‑κB signaling pathways to induce an overactive bladder in vivo

doi: 10.3892/etm.2024.12651

Figure Lengend Snippet: A HSD in vivo impaired barrier function of bladder. (A) H&E staining and histological score of bladder tissues (scale bar, 1 mm). Data were presented as the median with interquartile range. (B) Bladder weight/body weight ratio. (C) Thickness of lamina propria and mucosal layer of the bladder. Relative mRNA expression levels of (D) inflammatory response markers, IL-1β and TNF-α, (E) tight junction proteins, TJP-1 and Claudin-1 and (F) TRPV4. (G) Representative images of histological staining and quantification of protein expression of TRPV4, TJP-1 and CLAUDIN-1 in bladder tissues sections from CON and HSD mice. Scale bar, 250 µm (n=3). (H) Correlation analysis between the mRNA expression levels of tight junction proteins and inflammation factors in the bladder and urination characteristics in CON and HSD mice. Data are presented as mean ± SD (n=6). P-values were calculated using a two-tailed unpaired Student's t test; *** P<0.001, ** P<0.01 and * P<0.05. HSD, high salt diet; CON, control; TJP-1, tight junction protein 1; TRPV4, transient receptor potential vanilloid 4; ns, non-significant; A.U., arbitrary units.

Article Snippet: The samples were then incubated at 4˚C overnight with the following primary antibodies: Rabbit anti-TJP-1 antibody (1:250; cat. no. ab276131; Abcam), rabbit anti-CLAUDIN-1 antibody (1:200; cat. no. YT0942; ImmunoWay Biotechnology Company), rabbit anti-TRPV4 antibody (1:200; cat. no. YT5833; ImmunoWay Biotechnology Company), rabbit anti-CASP1 antibody (1:200; cat. no. YP0749; ImmunoWay Biotechnology Company) and rabbit anti-NF-κB antibody (1:200; cat. no. YM8001; ImmunoWay Biotechnology Company).

Techniques: In Vivo, Staining, Expressing, Two Tailed Test, Control

Journal: Experimental and Therapeutic Medicine

Article Title: A high salt diet impairs the bladder epithelial barrier and activates the NLRP3 and NF‑κB signaling pathways to induce an overactive bladder in vivo

doi: 10.3892/etm.2024.12651

Figure Lengend Snippet: A HSD increased uroepithelial oxidative stress in SV-HUC-1 cells. Relative mRNA expression levels of (A) TJP-1 and CLAUDIN-1, (B) TRPV4 and (C) IL-1β and TNF-α in HSD-treated and CON cells. (D) Relative MPO expression levels in CON and HSD groups. (E) Representative images and quantification of intracellular ROS levels (scale bar, 200 µm). (F) Relative mRNA expression levels of NCF-1 and CYBA. (G) Relative MDA expression levels in CON and HSD groups. Data are presented as mean ± SD (n=6). P-values were calculated using a two-tailed unpaired Student's t-test; *** P<0.001 and ** P<0.01. HSD, high salt diet; CON, control; SV-HUC-1, SV40 virus transformed human uroepithelium cells; TJP-1, tight junction protein 1; TRPV4, transient receptor potential vanilloid 4; MPO, myeloperoxidase; ROS, reactive oxygen species; NCF-1, neutrophil cytosolic factor 1; CYBA, cytochrome B-245 alpha chain; MDA, malondialdehyde.

Article Snippet: The samples were then incubated at 4˚C overnight with the following primary antibodies: Rabbit anti-TJP-1 antibody (1:250; cat. no. ab276131; Abcam), rabbit anti-CLAUDIN-1 antibody (1:200; cat. no. YT0942; ImmunoWay Biotechnology Company), rabbit anti-TRPV4 antibody (1:200; cat. no. YT5833; ImmunoWay Biotechnology Company), rabbit anti-CASP1 antibody (1:200; cat. no. YP0749; ImmunoWay Biotechnology Company) and rabbit anti-NF-κB antibody (1:200; cat. no. YM8001; ImmunoWay Biotechnology Company).

Techniques: Expressing, Two Tailed Test, Control, Virus, Transformation Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: (A) Time line of clinical course showing most significant clinical events, diagnostic studies, and therapeutic interventions. (B) Lumbosacral spine MRI, T2 weighted sequence, shows evidence of tethered cord with thickening of the filum terminale, but without evidence of intrathecal mass. (C) Plain film of the chest shows no evidence of skeletal dysplasia. (D) Plain film of the pelvis and hips shows no abnormalities. (E) Direct laryngoscopy demonstrates vocal cords fixed in a paramedian position and no abduction during inspiration. (F) Representation of TRPV4 protein domains and mutations with corresponding clinical phenotypes. The ligand binding site and the pore region are located between S2‐S3 and S5‐S6, respectively. Amino acids that are mutated in mixed phenotype TRPV4 channelopathies are indicated with an arrowhead and corresponding symbol (WT residue shown). Arginine 269 (underlined) is the most commonly reported mutation site in the literature. The novel R616G missense mutation (squared) affects a residue in transmembrane S5 helix that has been previously associated with skeletal dysplasia. Abbreviations: NCS, nerve conduction studies; PRD, proline‐rich domain; AR, ankyrin repeat; S1 to 6, transmembrane domains; TRP, transient receptor potential; MAP7, microtubule associated protein 7 binding site; CaM, calmodulin binding site. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Diagnostic Assay, Sequencing, Ligand Binding Assay, Mutagenesis, Binding Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: Nerve conduction studies in a 2‐year‐old patient with a novel TRPV4 R616G mutation.

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Mutagenesis

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: Systematic review of published literature of TRPV4 mutations associated with mixed neuropathy and skeletal dysplasia manifestations.

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Mutagenesis, Multiplex Assay, Ligand Binding Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: Expression and localization of selected TRPV4 mutants. (A) Cryo‐EM structure of the TRPV4 tetramer in an agonist‐bound state (PDB: 7AA5 ⁴⁷ ) depicting the location of mutated amino acid residues analyzed in vitro. Each monomer of the TRPV4 tetramer is displayed in a separate color. Colored spheres indicate the location of mutated amino acid residues within each monomer. (B) Immunoblot from HEK293T cells transfected with equal amounts of GFP‐tagged TRPV4 in the absence of TRPV4 antagonist. Expression of R616G and L619P appear reduced. (C) Immunoblot from HEK293T cells transfected with equal amounts of GFP‐tagged TRPV4 in the presence of 1 μM GSK219 TRPV4 antagonist. Expression of the individual mutants is normalized in the presence of antagonist. (D‐E) Immunohistochemistry of GFP‐tagged TRPV4 and actin (phalloidin) in MN‐1 cells (D) and MDCK cells (E) demonstrates normal trafficking to the cell membrane and co‐localization with cortical actin. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Expressing, Cryo-EM Sample Prep, In Vitro, Western Blot, Transfection, Immunohistochemistry

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: TRPV4 mutations analyzed and summary of in vitro results.

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: In Vitro, Ligand Binding Assay

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: Severe and mixed phenotype mutants cause marked elevations of baseline calcium levels. (A) Representative images from ratiometric calcium imaging experiments. MN‐1 cells were transfected with GFP‐tagged TRPV4 plasmids and loaded with Fura‐2 AM calcium indicator. Baseline and hypotonic‐stimulated calcium responses were then measured over time. Pathogenic TRPV4 mutants lead to elevated baseline calcium compared to WT TRPV4, whereas baseline calcium levels are more markedly elevated in R616G and L619P TRPV4. (B‐D) Averaged calcium imaging traces before and after hypotonic stimulation, denoted by vertical dashed line. (B) R269C and D333G TRPV4 cause elevated baseline and stimulated calcium influx compared to WT TRPV4. (C) R616G causes marked baseline calcium elevation, whereas R616Q causes only mild elevations. (D) S542Y and W785C TRPV4 cause larger increases in baseline calcium compared to R269C TRPV4. L619P causes marked elevation of baseline calcium similar to R616G. (E) Comparison of baseline calcium levels across all tested TRPV4 mutants. Brown‐Forsythe and Welch ANOVA with Dunnett's post‐hoc test, n = 11–32 independent experiments per condition. (F) Comparison of maximum hypotonic stimulated calcium levels across all tested TRPV4 mutants. One‐way ANOVA with Dunnett's post‐hoc test, n = 11–32 independent experiments per condition. (G) Comparison of calcium imaging traces from cells treated with 50 nM GSK219 and then subjected to hypotonic saline. The R616G and L619P mutants demonstrate retained responses to hypotonic saline stimulation. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Imaging, Transfection

Journal: Annals of Clinical and Translational Neurology

Article Title: TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

doi: 10.1002/acn3.51523

Figure Lengend Snippet: R616G and L619P cause increased cytotoxicity, reduced responsiveness to antagonism, and structural disruptions to the S5 transmembrane domain. (A) Cytotoxicity assay demonstrates increased toxicity with pathogenic mutants that is more pronounced with the R616G and L619P mutants. Repeated measures one‐way ANOVA with Dunnett's post‐hoc test, n = 4 independent experiments per condition. (B) Averaged intracellular calcium levels in TRPV4 mutants treated with escalating doses of GSK219 antagonist. (C) Inhibitory dose response curve comparing WT TRPV4 to R269C, D333G, and R616Q mutants. (D) Inhibitory dose response curve comparing WT TRPV4 to S542Y, R616G, L619P, and W785C mutants. (E) Location of R616 (red sphere) and L619 (blue sphere) in the S5 helix of TRPV4 shown on the cryo‐EM structure of the human TRPV4 channel in an agonist‐bound state (PDB: 7AA5 ⁴⁷ ). Each monomer of the TRPV4 tetramer is displayed in a separate color. On the right is a close‐up view of R616 (red) and L619 (blue) in the S5 helix as well as Y602 (orange) in the S4‐S5‐linker of a neighboring subunit with amino acid side chains shown in stick representations. R616 and Y602 side chain rotamers enabling a R616‐Y602 cation‐π interaction which presumably stabilizes the closed‐state were modeled as transparent sticks. In the structure of the agonist‐bound TRPV4 state, the R616 and Y602 side chains point away from each other such that the putative cation‐π interaction is prevented (opaque sticks). (F) Schematic representation of how the R616Q, R616G, and L619Q mutations may impact the TRPV4 structure. In the WT structure, the S5 helix and the R616‐Y602 cation‐π interaction are intact, whereas both are perturbed in the R616G and R616Q mutants. In the R616G mutant, the S5 helix is additionally perturbed due to the ability of glycine to act as a helix breaker. Likewise, substitution of proline in the L619P mutant can also structurally perturb the S5 helix. Breaking the S5 helix presumably disrupts the ion conducting pore and leads to uncontrolled ion flux. In contrast, the loss of the cation‐π interaction might lower the activation threshold by agonists. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Primary antibodies used were rabbit anti‐GFP (Cell Signaling Technology, 2555), mouse anti‐GFP (Thermo Fisher Scientific, A‐11120), rabbit anti‐TRPV4 (Cosmo Bio USA, KAL‐KM119).

Techniques: Cytotoxicity Assay, Cryo-EM Sample Prep, Mutagenesis, Activation Assay

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) Top: representative current-voltage relationships determined by whole-cell patch-clamp recordings of control and EPA (100 μM)-treated aortic endothelial cells challenged with GSK1016790A (GSK101, TRPV4 agonist, 0.1 μM) and GSK101 (0.1 μM) + HC067047 (HC, TRPV4 antagonist; 5 μM). Bckgrd. indicates background currents. Bottom: representative time course of whole-cell patch-clamp recordings (+80 mV) of control and EPA-treated aortic endothelial cells challenged with GSK101 and inhibited with HC. (B) Boxplots show the mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of TRPV4 currents ((I GSK101 – I HC ) pA/pF) obtained by whole-cell patch-clamp recordings (+80 mV) of control and EPA-treated endothelial cells from skin, retina, lung, brain, and aorta. Two-way ANOVA and Sidak-Holm multiple comparisons test. (C) Left: representative current-voltage relationships determined by whole-cell patch-clamp recordings of control and EPA (100 μM)-treated aortic endothelial cells challenged with GSK101 (from 1 to 2,000 nM). Currents evoked by GSK101 submaximal concentrations (gray and red traces) were normalized by corresponding currents elicited by saturating GSK101 (2,000 nM; black traces) per cell. Right: normalized (I/I max ) GSK101 dose-response profiles of control and EPA (100 μM)-treated aortic endothelial cells. A Hill function was fitted to the data. The shadows encompassing the curves indicate the 95% confidence bands for the fit. Circles are mean ± SD. n = 36 for control and n = 36 for EPA (100 μM)-treated aortic endothelial cells. (D) Top: representative western blots (anti-TRPV4) of the membrane fractions of control and EPA (100 μM)-treated human endothelial cells from skin, retina, lung, brain, and aorta. Bottom: mean/scatter-dot plot showing relative intensities of TRPV4 bands, calculated from total protein detection of chemically labeled proteins (Stain-Free System Bio-Rad), from the membrane fractions of control and EPA (100 μM)-treated endothelial cells. Lines are mean ± SD. Two-way ANOVA. Asterisks indicate values significantly different from control (***p < 0.001 and **p < 0.01) and n.s. indicates values not significantly different from the control. n is indicated in each panel. See also and .

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Patch Clamp, Western Blot, Labeling, Staining

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) The C. elegans fatty acid desaturase FAT-1 enzyme introduces a double bond in ω -6 arachidonic acid to synthesize ω -3 EPA in worms and transgenic fat-1 mice, but not in WT mice. Mice and C. elegans cartoons were created with BioRender.com . (B) Gardner-Altman estimation plot showing the mean difference in EPA-membrane content of whole mesenteric arteries of WT and fat -1 mice, as determined by LC-MS. The raw data are plotted on the left axis. The mean difference, on the right, is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bars. Mann-Whitney rank test for two independent groups. (C) Representative current-voltage relationships determined by whole-cell patch-clamp recordings of WT and fat -1 cultured isolated mesenteric endothelial cells challenged with GSK101 (0.1 μM) and GSK101 (0.1 μM) + HC (10 μM). Bckgrd. indicates background currents. (D) Bar graph displaying TRPV4 currents ((I GSK101 – I HC ) pA/pF) obtained by whole-cell patch-clamp recordings (+80 mV) of cultured isolated mesenteric endothelial cells of WT and fat -1 mice. Bars are mean ± SEM. Two-tailed unpaired t test. Asterisks indicate values significantly different from WT (**p < 0.01 and *p < 0.05). n is indicated in each panel.

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Transgenic Assay, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY, Patch Clamp, Cell Culture, Isolation, Two Tailed Test

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) Representative time course of GSK101 (5 nM)-induced vasodilation of pressurized (80 mmHg) mesenteric arteries from WT and fat-1 mice. Inset: micrograph of a representative cannulated mesenteric artery. (B) Percentage of GSK101 (5 nM)-induced vasodilation of mesenteric arteries (endothelium-intact or -denuded) from WT and fat-1 mice. Bars are mean ± SEM. Two-way ANOVA and Tukey multiple comparisons test. (C) Representative western blot (anti-TRPV4) of the membrane fractions of WT and fat-1 mice mesenteric arteries. (D) Mean/scatter-dot plot showing relative intensities of TRPV4 bands, calculated from total protein detection of chemically labeled proteins (Stain-Free System Bio-Rad), from the membrane fractions of mesenteric arteries from WT and fat-1 mice. Lines are mean ± SD. Two-tailed unpaired t test. Asterisks indicate values significantly different from WT (**p < 0.01) and n.s. indicates values not significantly different from the WT. n is indicated in each panel. See also .

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Western Blot, Labeling, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) Gardner-Altman estimation plot showing the mean difference in EPA-membrane content of whole mesenteric arteries of WT mice fed with standard or ω -3 fatty acid-enriched diets, as determined by LC-MS. The raw data are plotted on the left axis. The mean difference, on the right, is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bars. Mann-Whitney rank test for two independent groups. Mouse cartoon was created with BioRender.com . (B) Representative time course of GSK101 (5 nM)-induced vasodilation of pressurized (80 mmHg) mesenteric arteries from WT mice fed with standard or ω -3 fatty acid-enriched diets. (C) Percentage of GSK101 (5 nM)-induced vasodilation of mesenteric arteries from WT mice fed with standard or ω -3 fatty acid-enriched diets. Bars are mean ± SEM. Two-tailed unpaired t test. (D) Boxplots show the mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of the percentage of maximal GSK101 (5 nM)-induced vasodilation remaining at the time GSK101 was removed from the mesenteric arteries of WT mice fed with standard or ω -3 fatty acid-enriched diets. Two-tailed unpaired t test. (E) Top: representative western blot (TRPV4) from membrane fractions of the mesenteric arteries from WT mice fed with standard or ω -3 fatty acid-enriched diets. Bottom: mean/scatter-dot plot showing relative intensities of TRPV4 bands, calculated from total protein detection of chemically labeled proteins (Stain-Free System Bio-Rad), from the membrane fractions of mesenteric arteries from WT mice fed with standard or ω -3 fatty acid-enriched diets. Lines are mean ± SD. Two-tailed unpaired t test. (F) Representative current-voltage relationships determined by whole-cell patch-clamp recordings of cultured isolated mesenteric endothelial cells, from WT mice fed with standard or ω -3 fatty acid-enriched diets, challenged with GSK101 (0.1 μM) and GSK101 (0.1 μM) + HC (10 μM). (G) Bar graph displaying TRPV4 currents ((I GSK101 – I HC ) pA/pF) obtained by whole-cell patch-clamp recordings (+80 mV) of cultured isolated mesenteric endothelial cells of WT mice fed with standard or ω -3 fatty acid-enriched diets. Bars are mean ± SEM. Two-tailed unpaired t test. (H) Top: representative time course of sodium nitroprusside (SNP) (1 μM)-induced vasodilation of pressurized (80 mmHg) mesenteric arteries from WT mice fed with standard or ω -3 fatty acid-enriched diets. Bottom: percentage of SNP (1 μM)-induced vasodilation of WT mice fed with standard or ω -3 fatty acid-enriched diets. Bars are mean ± SEM. Two-tailed unpaired t test. Asterisks indicate values significantly different from standard diet (***p < 0.001, **p < 0.01, and *p < 0.05) and n.s. indicates values not significantly different from the standard diet. n is indicated in each panel. See also .

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY, Two Tailed Test, Western Blot, Labeling, Staining, Patch Clamp, Cell Culture, Isolation

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) Representative time course of whole-cell patch-clamp recordings (−60 mV) of control and EPA (100 μM)-treated human aortic endothelial cells challenged with GSK101 (1 μM). (B) Bar graph displaying TRPV4 currents (pA/pF) obtained by whole-cell patch-clamp recordings (−60 mV) of control and EPA (100 μM)-treated aortic endothelial cells. Bars are mean ± SEM. Mann-Whitney rank test for two independent groups. (C) Boxplots show mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of the time to reach half amplitude from the peak current (Imax) elicited by GSK101 (1 μM) in control and EPA (100 μM)-treated aortic endothelial cells. Two-tailed unpaired t test. (D) Micrographs of cultured isolated mesenteric endothelial cells from WT mice fed with standard or ω -3 fatty acid-enriched diets, loaded with Fluo-4 AM, and challenged with GSK101 (0.1 μM). The color bar indicates a relative change in fluorescence intensity. Experiments were performed in three independent cell preparations. t indicates the times at which representative micrographs were taken from the traces in (E). (E) Representative traces corresponding to normalized intensity changes (ΔF/F) of individual cells shown in (D). (F) Area under the curve (AUC) of the fluorescence response (ΔF/F), depicted as violin plots with the means shown as horizontal bars, of endothelial cells from WT mice fed with standard or ω -3 fatty acid-enriched diets challenged with GSK101. Two-tailed unpaired t test. (G) Micrographs of cultured isolated mesenteric endothelial cells of WT mice fed with standard or ω -3 fatty acid-enriched diets, loaded with Fluo-4 AM and challenged with a hypoosmotic buffer (HB: 240 mOsm). The color bar indicates a relative change in fluorescence intensity. Experiments were performed in two independent cell preparations. t indicates the times at which representative micrographs were taken from the traces in (H). (H) Representative traces corresponding to normalized intensity changes (ΔF/F) of individual cells shown in (G). (I) AUC of the fluorescence response (ΔF/F), depicted as violin plots with the means shown as horizontal bars, of endothelial cells from WT mice fed with standard or ω -3 fatty acid-enriched diets challenged with a hypoosmotic buffer. Two-tailed unpaired t test. Asterisks indicate values significantly different from control or standard diet (***p < 0.001 and **p < 0.01). n is indicated in each panel.

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Patch Clamp, MANN-WHITNEY, Two Tailed Test, Cell Culture, Isolation, Fluorescence

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: (A) Cartoon depicting the full-length rat TRPV4 channel. (B) Representative time course of whole-cell patch-clamp recordings (−60 mV) of control and EPA (100 μM)-treated HEK293 cells, expressing full-length rat TRPV4, challenged with GSK101 (1 μM). Traces were normalized for comparison. (C) Boxplots show mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of the time to reach half amplitude from the peak current (Imax) elicited by GSK101 (1 μM) in control and EPA (100 μM)-treated HEK293 cells expressing full-length rat TRPV4. Two-tailed unpaired with Welch’s correction. (D) Cartoon depicting the Δ186 rat TRPV4 channel construct. (E) Representative time course of whole-cell patch-clamp recordings (−60 mV) of control and EPA (100 μM)-treated HEK293 cells, expressing the Δ186 rat TRPV4 channel construct, challenged with GSK101 (1 μM). Traces were normalized for comparison. (F) Boxplots show mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of the time to reach half amplitude from the peak current (Imax) elicited by GSK101 (1 μM) in control and EPA (100 μM)-treated HEK293 cells expressing the Δ186 rat TRPV4 channel construct. Two-tailed unpaired t test. (G) Cartoon depicting the 5Ala (K121A, R122A, R124A, R125A, K126A) rat TRPV4 channel construct. (H) Representative time course of whole-cell patch-clamp recordings (−60 mV) in control and EPA (100 μM)-treated HEK293 cells expressing the 5Ala rat TRPV4 channel construct challenged with GSK101 (1 μM). Traces were normalized for comparison. (I) Boxplots show mean (gray circle), median (bisecting line), SD (whiskers), and SEM (box) of the time to reach half amplitude from the peak current (Imax) elicited by GSK101 (1 μM) of control and EPA (100 μM)-treated HEK293 cells expressing the 5Ala rat TRPV4 channel construct. Two-tailed unpaired t test. Asterisks indicate values significantly different from control (**p < 0.01) and n.s. indicates values not significantly different from the control. n is indicated in each panel. See also .

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Patch Clamp, Expressing, Two Tailed Test, Construct

Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-human TRPV4 , Abcepta , Cat# AP18990a, RRID: AB_2923215.

Techniques: Recombinant, Protease Inhibitor, Staining, Western Blot, Protein Extraction, Plasmid Preparation, Software, Imaging, Microscopy

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Quantification of TRPV4 expression. (a) Sites where tissue samples were taken and the quantification method for stained areas. Tissue was collected from 57 patients who underwent gastric resection. The following areas were sectioned in all layers: 1. greater curvature of the fornix; 2. greater curvature of the gastric body; 3. greater curvature of the antrum; 4. anterior wall of the gastric body; 5, posterior wall of the gastric body. (b) TRPV4 immunostaining in gastric tissue. (c) The images were captured using a microscope with BZ-X710 (Keyence, Osaka, Japan), and staining was quantified with hybrid cell count BZ-H4C.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Expressing, Staining, Immunostaining, Microscopy, Cell Counting

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Comparison of stained areas between nonobese and obese cases. (a) Renal tissues were used as a positive control for TRPV4-immunostaining. (b) For the negative control, tissues were subjected to immunostaining without the primary antibody. (c) Immunostaining of the nonobese and obese stomach. (d) Comparison of stained areas in tissue samples collected from patients with and without obesity. Lines within the boxes represent median values; upper and lower lines of the boxes represent 25th and 75th percentiles, respectively; upper and lower bars outside the boxes represent the maximum and minimum, respectively ( ∗∗ p < 0.01).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Comparison, Staining, Positive Control, Immunostaining, Negative Control

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: TRPV4 immunostaining in the stomach of patients with and without obesity.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Immunostaining

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Fluorescence immunostaining of TRPV4 in MGN3-1 cells. TRPV4 protein expression was observed in MGN3-1 cells. Magnification: ×600. TRPV4-mediated change in [Ca 2+ ] i in MGN3-1 cells.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Fluorescence, Immunostaining, Expressing

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: TRPV4-mediated changes in cytosolic Ca 2+ in MGN3-1 cells. The effects of GSK1016790 A and HC 067047 on [Ca 2+ ] i in MGN3-1 cells. Compared to the control level at 180 s, [Ca2+] i increased in MGN3-1 cells treated with GSK1016790 A ( n = 20, p < 0.01) but decreased in cells treated with GSK1016790 A and HC067047 ( n = 20, p < 0.01) (GSK1016790 A, TRPV4 agonist; HC067047, TRPV4 antagonist).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Control

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Regulation of ghrelin secretion by the TRPV4 agonist and antagonist in MGN3-1 cells. (a) The amount of secreted acyl ghrelin after four-hour treatment of MGN3-1 cells with GSK1016790 A (3 μ M) or HC067047 (20 μ M) + GSK1016790 A (3 μ M). (b) The amount of secreted des-acyl ghrelin after four-hour treatment of MGN3-1 cells with GSK1016790 A (3 μ M) or HC067047 (20 μ M) + GSK1016790 A (3 μ M) ( ∗ p < 0.05; ∗∗ p < 0.01; n = 6; error bars: standard error of the mean).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: