trpv4 antagonist hc 067 047  (MedChemExpress)

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A, B ) Five-day TGFβ2 treatment (1 ng/ml) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. ( C ) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4–8 experiments, each gene tested in 3–7 different pTM strains (see ). Two-tailed one-sample t -test of TGFβ2-induced gene expression levels as a percent of control samples. ( D ) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. *p < 0.05, **p < 0.01. Figure 1—source data 1. Uncropped images of the membrane and HRP-signal for the western blots shown in (labelled). Figure 1—source data 2. Uncropped images of the membrane and HRP-signal for the western blots shown in .

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Expressing, Two Tailed Test, Gene Expression, Control, Isolation, Membrane, Western Blot

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Five-day TGFβ2 treatment (1 ng/ml) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca 2+ influx in primary trabecular meshwork (pTM) cells compared to serum-free media alone treated cells tested on the same day ( N = 5 pTM strains, n = 3–5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one-sample t -test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. ( B ) Violin plots showing the distribution of GSK101-induced Ca 2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ( C ) Representative traces showing TRPV4 agonist-induced Ca 2+ influx (seen as an increase in F 340 / F 380 ) in pTM (mean ± SEM of 4 representative cells/group), alongside example Fura-2-loaded pTM cells before ( i ), during ( ii ), and after ( iii ) GSK101 application. Scale bar = 50 µm. **p < 0.01.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Two Tailed Test, Control

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) TGFβ2 treatments for 24 hr at 1 ng/ml ( N = 6 pTM strains, n = 3–5 slides/condition/day) or 5 ng/ml ( N = 5 pTM strains, n = 3–5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca 2+ influx and were significantly lower than cells treated with TGFβ2 for 5 days at 1 ng/ml (5 days TGFβ2 results from ). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1 day treatment groups compared to control groups shown in . ( B ) Representative traces for GSK101 response following 24 hr TGFβ2 treatment, traces show mean ± SEM of 3–4 cells. ( C ) Average current density in response to GSK101 (24 hr control: n = 11 cells, 24 hr TGFβ2: n = 10 cells) shows generally increased current in TGFβ2-treated cells. Data show mean ± SEM ( D, E ). Violin plots of individual cell strains shown in A . Thick dashed line indicates mean, while light dashed line indicates quartiles. ** p < 0.01, *** p < 0.001.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Control

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Intravitreal injection of LV-TGFβ2 (Week 1), but not LV-Control, elevates intraocular pressure (IOP) in WT mice ( N = 5 eyes/group) as early as 1-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2-treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post hoc analysis ( B ) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A . Two-way ANOVA with Bonferroni post hoc analysis. ( C ) Intravitreal injection of LV-TGFβ2 in Trpv4 −/− mice ( N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A, B, N = 8–15 eyes/group). ( D ) Statistical comparison of the IOP values shown in C . The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4 −/− eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4 −/− eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post hoc analysis. ( A, C ) shows mean ± SEM. Data in ( B, D ) show individual data points over mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 5—source data 1. Source data for Lv-Control IOP and Lv-TFFb2 cohorts treated with HC-06. Figure 5—source data 2. Source data for IOP data from WT and Trpv4 KO eyes treated with TGFβ2.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Injection, Control, Produced, Comparison

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Intraocular pressure (IOP) in LV-TGFβ2-injected eyes was significantly elevated compared to both LV-Ctrl injected WT and Trpv4 −/− eyes, as well as LV-TGFβ2-injected Trpv4 −/− eyes ( N = 6 eyes/condition).

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Injection

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca 2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate the Rho/ROCK pathway to augment cytoskeletal contractility and stimulate extracellular matrix (ECM) release. Actomyosin contractility promotes outflow resistance and drives OHT and underpins a vicious feedforward TRPV4-dependent loop that maintains OHT. This figure was created using BioRender.com .

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Functional Assay

Journal: Nature Communications

Article Title: Fallopian tube rheology regulates epithelial cell differentiation and function to enhance cilia formation and coordination

doi: 10.1038/s41467-024-51481-9

Figure Lengend Snippet: a Representative widefield images of live ciliated cells, and the corresponding ( b ) phase angle, ( c ) wave direction and ( d ) wavelength of beating cilia as a function of media viscosity. Scale bars, 10 µm. e Average cilia beating frequency, and f coherence (a measure of the cilia coordination) as a function of culture media viscosity and for cells treated with RN-1734 to block the TRPV4 channel, and for the vehicle control. Data are represented as mean ± s.d. and analysed using one-way ANOVA with Tukey post hoc testing, * P ≤ 0.05, ** P ≤ 0.01 and **** P ≤ 0.0001, n = 9 images from three biological replicates per each condition. For exact P values see Supplementary Table . Source data are provided as a Source Data File.

Article Snippet: For TRPV4 inhibition experiment, cells were incubated with 10 µM of TRPV4 antagonist RN-1734 (Sigma-Aldrich, R0658) dissolved in DMSO prior to experiments.

Techniques: Viscosity, Blocking Assay, Control

Journal: Nature Communications

Article Title: Fallopian tube rheology regulates epithelial cell differentiation and function to enhance cilia formation and coordination

doi: 10.1038/s41467-024-51481-9

Figure Lengend Snippet: a Calcium uptake expressed as the ratio of F/F 0 over 120 s for cells treated with control or RN-1734 (to block the TRPV4 channel) as compared with a control group of untreated cells. F is the instantaneous fluorescence intensity and F 0 is the baseline intensity. b The zoomed-in view for a low MMP cell with JC-1 monomer (top) and a high MMP cell with JC-1 aggregate (bottom). Scale bar, 10 µm. c Representative overlaid immunofluorescence staining images of epithelial cells stained with JC-1 at 72-h time point as a function of culture media viscosity. JC-1 monomers and aggregates were imaged in green and red channels, respectively. The asterisks ( * ) indicate cells with low mitochondrial membrane potential (MMP). Cell nuclei were stained using blue-fluorescent Hoechst 33342. Scale bar, 20 µm. d The ratio of low MMP (green) to high MMP (red) cells as a function of culture media viscosity, indicating the degree of depolarization. Experiments were carried out on n = 3 from three biological replicates and ≥ 200 cells for each condition were analysed. e TRPV4 fluorescence intensity (TRPV4 activated channels) in cells as a function of culture media viscosity, n ≥ 6 images from three biological replicates per each condition. All data are represented as mean ± s.d. and analysed using one-way ANOVA with Tukey post hoc testing, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. For exact P values see Supplementary Table . Source data are provided as a Source Data File.

Article Snippet: For TRPV4 inhibition experiment, cells were incubated with 10 µM of TRPV4 antagonist RN-1734 (Sigma-Aldrich, R0658) dissolved in DMSO prior to experiments.

Techniques: Control, Blocking Assay, Fluorescence, Immunofluorescence, Staining, Viscosity, Membrane