Journal: CNS Neuroscience & Therapeutics

Article Title: Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia

doi: 10.1111/cns.12494

Figure Lengend Snippet: Immunoreactivity (IR) of TRPV4 in focal cortical dysplasia (FCD) types Ia (FCDIa), IIa (FCDIIa), and IIb(FCDIIb). (A–C) TRPV4 IR in normal control cortex (CTX). Weak‐to‐moderate TRPV4 in neurons (arrows in [A] and [B]), glial cells (arrowheads in [B] and [C]) and weak TRPV4 in endothelial cells (double arrows in [C]) in neocortex, white matter(WM), and junction. (D–F) TRPV4 IR in FCDIa. Moderate‐to‐strong TRPV4 in neurons, including in microcolumns ([D], inset b in [D]) and in endothelial cells (double arrows in inset a in [D]), and moderate TRPV4 IR in glial cells (arrowheads in insert a in [D]). (E, F) Merged images show the colocalization of TRPV4 (green) with NeuN (red) in neurons (arrows in [E]) and the colocalization of TRPV4 (green) with glial fibrillary acidic protein ([GFAP] red) in glial cells (arrowheads) but not in neurons (arrows in [F]). (G–I) TRPV4 IR in FCDIIa. Moderate‐to‐strong TRPV4 IR in dysmorphic neurons(DNs) (arrows in [G]) and in endothelial cells ([G] inset); strong TRPV4 IR in glial cells (arrowheads in [G]). The merged images show the colocalization of TRPV4 (green) with NF200 (red) in DNs(arrows in H). (I) TRPV4 (green) colocalized with GFAP(red) in glial cells (arrowheads in [I]) but not in DNs (arrows in [I]). (J‐L) TRPV4 IR in FCDIIb. Moderate‐to‐strong TRPV4 IR in endothelial cells (double arrows in [J]), DNs (arrows in [J]), and ballon cells ([BCs], double arrowheads in [J]) with different sizes and shape. There is strong TRPV4 IR in glial cells (arrowheads in [J] and [R]). (K) Confocal image indicated that TRPV4‐positive DNs (arrows) and BCs (triple arrowheads) colocalize with NF200 (red); other BCs (double arrowheads) do not colocalize with NF200 (red). (L) Merged image shows that TRPV4‐positive glial cells (inset a in [L], green) and TRPV4‐positive BCs (double arrowheads in [L] and inset b, green) colocalize with GFAP (red); other BCs (triple arrowheads, inset b in [L]) do not colocalize with GFAP. Sections are counterstained with hematoxylin (A–D, G, J) or DAPI ([E], [F], [H], [I], [K], [L], inset in [L]). Scale bars = (A–D, G, J) 50 μm; (E, F, H, I, K, L) 30 μm.

Article Snippet: Then, the membranes were blocked in 5% nonfat dry milk for 2 h and incubated overnight at 4°C with one of the following primary antibodies: TRPV4 (rabbit polyclonal, 1:800; Alomone, Jerusalem, Israel), PKC (mouse monoclonal, 1:800; Boster, China), PKA (rabbit polyclonal, 1:1000; Proteintech, Wuhan, China), and GAPDH (rabbit monoclonal, 1:1000; Cell Signaling, Beverly, MA, USA).

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia

doi: 10.1111/cns.12494

Figure Lengend Snippet: Alteration in TRPV4 expression in focal cortical dysplasia (FCD).(A, B) Representative immunoblot bands (A) and densitometricanalyses (B) of total homogenates from FCD types Ia(FCDIa), IIa (FCDIIa) and IIb (FCDIIb) lesions and normal control cortex (CTX) tissue samples. *P < 0.05, **P < 0.01, # P > 0.05, ANOVA. OD = optical densities. Number of samples is indicated in columns for each condition.

Article Snippet: Then, the membranes were blocked in 5% nonfat dry milk for 2 h and incubated overnight at 4°C with one of the following primary antibodies: TRPV4 (rabbit polyclonal, 1:800; Alomone, Jerusalem, Israel), PKC (mouse monoclonal, 1:800; Boster, China), PKA (rabbit polyclonal, 1:1000; Proteintech, Wuhan, China), and GAPDH (rabbit monoclonal, 1:1000; Cell Signaling, Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia

doi: 10.1111/cns.12494

Figure Lengend Snippet: Staining Scores of TRPV4‐, PKC‐, PKA‐Immunopositive Cells in FCD and Control Specimens

Article Snippet: Then, the membranes were blocked in 5% nonfat dry milk for 2 h and incubated overnight at 4°C with one of the following primary antibodies: TRPV4 (rabbit polyclonal, 1:800; Alomone, Jerusalem, Israel), PKC (mouse monoclonal, 1:800; Boster, China), PKA (rabbit polyclonal, 1:1000; Proteintech, Wuhan, China), and GAPDH (rabbit monoclonal, 1:1000; Cell Signaling, Beverly, MA, USA).

Techniques: Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia

doi: 10.1111/cns.12494

Figure Lengend Snippet: Function assessment of TRPV4 expression in primary cortical neurons. (A) Representative phase contrast images show cultured rat cortical neurons. (B–D) Merged images show the colocalization of TRPV4 (green) with MAP2 (red) in cultured cortical neurons. The nuclei are stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI) and are shown in blue. (E–F) Confocal images show primary cultured cortical neurons labeled with Fluo‐3 before ([E]) and after ([F]) 4α PDD treatment. (G) Dose–response curve indicated that dose‐dependent activation of Ca2 + influx under 4α PDD stimulation. Cultured cortical neurons were stimulated with 4α PDD at 3, 5, 10, 20, 30, and 100 μM. The dose–response curve was fitted by a sigmoidal dose–response function using GraphPad Prism 6.0. (H) Representative example showing the effect of TRPV4 agonist and antagonist, 4α PDD and HC067047, on [Ca2+]i in cultured cortical neurons. (I) Representative example showing removal of extracellular Ca2+ abolishing the 4α PDD response. The changes of intracellular calcium concentration in all experiments measured as fluo‐3 AM fluorescence were expressed relative to the basal level (F/F0). Scale bars: 30 μm.

Article Snippet: Then, the membranes were blocked in 5% nonfat dry milk for 2 h and incubated overnight at 4°C with one of the following primary antibodies: TRPV4 (rabbit polyclonal, 1:800; Alomone, Jerusalem, Israel), PKC (mouse monoclonal, 1:800; Boster, China), PKA (rabbit polyclonal, 1:1000; Proteintech, Wuhan, China), and GAPDH (rabbit monoclonal, 1:1000; Cell Signaling, Beverly, MA, USA).

Techniques: Expressing, Cell Culture, Staining, Labeling, Activation Assay, Concentration Assay, Fluorescence

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ablation of PM20D1 reveals N -acyl amino acid control of metabolism and nociception

doi: 10.1073/pnas.1803389115

Figure Lengend Snippet: Antagonism of TRP channels by PM20D1-regulated C18:1-Gln. ( A ) Change in relative Fluo-4 fluorescence (∆F/F 0 ) over time in cells transfected with TRPV1 (RFP + ) or in neighboring untransfected cells (RFP − ) following treatment with capsaicin. ( B ) Agonist response (∆F/F 0 ) in TRPV1-transfected cells after treatment with capsaicin or the indicated NAA (50 µM). ( C – E ) Antagonism of capsaicin-induced ∆F/F 0 by the indicated compound (50 µM) in TRPV1-transfected cells. ( F ) Dose response of C18:1-Gln antagonizing capsaicin-induced ∆F/F 0 in TRPV1-transfected cells. ( G – I ) Antagonism of agonist-induced ∆F/F 0 by the indicated compound (50 µM) in TRPV3- ( G ), TRPV4- ( H ), or TRPA1- ( I ) transfected cells. For A – I , transfection was performed in HEK293A cells. For C – I , antagonist studies were performed by preincubating the indicated compound for 2 min. Agonist-induced ∆F/F 0 was then quantified 1 min after addition of capsaicin (10 µM) or AITC (10 µM) or 12 s after addition of 2-ABP (500 µM) or GSK1016790A (0.2 µM). Data are shown as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 for the indicated comparison, or versus the corresponding DMSO control group. Fifteen to thirty cells were quantified per data point.

Article Snippet: Plasmids were obtained from the following sources: C-terminally tagged mouse TRPV1 (catalog no. MR227160; OriGene), C-terminally tagged human TRPV3 (catalog no. RC211184; OriGene); C-terminally tagged rat TRPV4 (plasmid no. 45751; Addgene); and pTurboRFP-Mito (catalog no. FP237; Evrogen).

Techniques: Fluorescence, Transfection, Comparison, Control

Journal: bioRxiv

Article Title: Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

doi: 10.1101/2023.05.26.542500

Figure Lengend Snippet: For (a-b), confluent monolayers of GCaMP transfected HAECs were exposed to laminar shear stress (20 dynes/cm 2 ) for 48 h prior to live cell imaging. a, Representative GCaMP intensity trace showing Ca 2+ activity at the downstream tip after the addition of EGTA (1.6 µM) to chelate calcium ions in the culture media. b, Representative GCaMP intensity trace showing Ca 2+ activity at the downstream tip after the addition of the Trpv4 antagonist GSK205 (20 µM). For (c-d), HAECs were seeded on y-shaped slides and exposed to unidirectional laminar flow for 48 h prior to staining. Immunofluorescence was compared for cells in low-flow (∼5 dynes/cm 2 ) and high-flow regions (∼20 dynes/cm 2 ). c, Trpv4 protein staining showed no difference for low flow versus high flow regions (representative images from n = 3 biological replicates; statistics calculated by two-tailed, unpaired t test to show no significance, ns, between regions). Quantifying the subcellular distribution of expression indicates that Trpv4 was not polarized under flow. Shown are means + SD from 44 low-flow and 57 high-flow cells. d, Representative images of ZO-1 staining (green) and proximity ligation assay (PLA) to detect Trpv4 and Caveolin-1 association (magenta puncta) in low-flow and high-flow regions. Cells in the high-flow region showed a significant increase in PLA puncta per cell compared to cells in the low-flow region. Shown are puncta/cell as well as mean ± SD and statistics calculated using unpaired, two-tailed t test; **** P < 0.0001. Additional segmentation analysis showed that Trpv4 and Caveolin-1 PLA puncta preferentially occurred in the downstream end. 111 cells were analyzed. Data was analyzed by one-way ANOVA and post hoc Tukey’s multiple comparisons test; **** P < 0.0001 (Upstream vs. Downstream; Mid vs. Downstream).

Article Snippet: Primary antibodies used: Erg (Abcam #ab92513), Ve-cadherin (BD Pharmingen™ #550547), Caveolin-1 (Invitrogen #PA1-064), Caveolin-1 (R&D AF5736), Caveolin-1 (Santa Cruz #sc-894), Cavin-1 (Abcam #ab48824), Icam-1 (Santa Cruz #sc-107), E-selectin (Invitrogen #MA1-06506), ZO-1 (Invitrogen #MA3-39100-A488), NFkB p65 (Cell Signaling #8242S), p-eNOS Ser1177 (Invitrogen #PA5-35879), p-eNOS Ser1177 (Santa Cruz #sc-81510), Trpv4 (Alomone Labs #ACC-034 or LSBio #LS-C401108), VE-Cadherin (R&D #AF938).

Techniques: Transfection, Live Cell Imaging, Activity Assay, Staining, Immunofluorescence, Two Tailed Test, Expressing, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

doi: 10.1101/2023.05.26.542500

Figure Lengend Snippet: a, Heat map displaying Ca 2+ activity for cells in the presence of high laminar flow before and after treatment with EGTA (1.6 mM). b, Heat map showing the subcellular location of Ca 2+ activity for cells under high laminar flow before and after treatment with GSK205 (20 µM). c, Gene expression analysis in HAECs cultured for 48 h in static or flow conditions shown as mean mRNA relative abundance ± SD (n = 3 biological replicates per condition). Note the flow-induced expression of KFL2 and KLF4 but unchanged expression for TRPV4. d, Western blot analysis of eNOS, Trpv4, Cav1 and GAPDH in HAECs cultured for 48 h under static or flow (20 dynes/cm 2 ) conditions. Protein expression was determined by densitometry, normalized to GAPDH levels and then expressed as means ± SD (n = 3 biological replicates per condition). ns, not significant, *** P < 0.001 by two- tailed, unpaired t test. e, Confocal images of representative controls for the PLA. Strong PLA was observed for Caveolin-1/Cavin-1. No PLA puncta were observed for Caveolin-1 and nuclear HistoneH3 or when Caveolin-1 or Trpv4 were used on their own. f, Quantification of the PLA for Trpv4/Caveolin-1 in HAECs in low-flow regions (as described for ). Shown are means ± SD (n = 49 cells analyzed) with statistics calculated using one-way ANOVA. ns, not significant.

Article Snippet: Primary antibodies used: Erg (Abcam #ab92513), Ve-cadherin (BD Pharmingen™ #550547), Caveolin-1 (Invitrogen #PA1-064), Caveolin-1 (R&D AF5736), Caveolin-1 (Santa Cruz #sc-894), Cavin-1 (Abcam #ab48824), Icam-1 (Santa Cruz #sc-107), E-selectin (Invitrogen #MA1-06506), ZO-1 (Invitrogen #MA3-39100-A488), NFkB p65 (Cell Signaling #8242S), p-eNOS Ser1177 (Invitrogen #PA5-35879), p-eNOS Ser1177 (Santa Cruz #sc-81510), Trpv4 (Alomone Labs #ACC-034 or LSBio #LS-C401108), VE-Cadherin (R&D #AF938).

Techniques: Activity Assay, Expressing, Cell Culture, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

doi: 10.1101/2023.05.26.542500

Figure Lengend Snippet: a, MβCD was used to deplete plasma membrane cholesterol. b, Flow-aligned HAECs were treated with MβCD for 30 min then fixed and stained for Caveolin-1 and DAPI. MβCD treatment abolished Caveolin-1 polarization as clearly shown by intensity plots of representative cells from the control and MβCD treated groups. c, NO production was visualized via DAF-FM loaded flow-aligned monolayers of control and MβCD treated groups. Shown are mean DAF-FM fluorescence intensities ± SD for n = 3 biological replicates and statistics calculated using two-tailed, unpaired t test; ** P = 0.0044. d, GCaMP imaging of the cholesterol-depleted cells under flow (20 dynes/cm 2 ) for 20 min showed lack of Ca 2+ activity. Displayed are time-dependent images of a representative cell and the corresponding intensity trace for the whole cell and indicated segments. At t = 20 min (blue arrow), the Trpv4 agonist GSK1016709A (GSK101, 10 nM) was added to the flowing culture media. This led to an immediate Ca 2+ burst as seen in the image at 20.1 min. e, Overall, only 13% of the cells depleted for cholesterol were active in the initial 20 min of imaging. The number of active cells increased to 75% following the addition of GSK101. f, IoD heatmaps show Ca 2+ activity following cholesterol depletion and subsequent GSK101 addition for n = 244 cells.

Article Snippet: Primary antibodies used: Erg (Abcam #ab92513), Ve-cadherin (BD Pharmingen™ #550547), Caveolin-1 (Invitrogen #PA1-064), Caveolin-1 (R&D AF5736), Caveolin-1 (Santa Cruz #sc-894), Cavin-1 (Abcam #ab48824), Icam-1 (Santa Cruz #sc-107), E-selectin (Invitrogen #MA1-06506), ZO-1 (Invitrogen #MA3-39100-A488), NFkB p65 (Cell Signaling #8242S), p-eNOS Ser1177 (Invitrogen #PA5-35879), p-eNOS Ser1177 (Santa Cruz #sc-81510), Trpv4 (Alomone Labs #ACC-034 or LSBio #LS-C401108), VE-Cadherin (R&D #AF938).

Techniques: Staining, Fluorescence, Two Tailed Test, Imaging, Activity Assay

Journal: bioRxiv

Article Title: Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

doi: 10.1101/2023.05.26.542500

Figure Lengend Snippet: a, HAEC monolayers were flow aligned for 48 h followed by the addition of DMSO (control) or Trpv4 antagonist GSK205 (20 µM) in the presence of laminar flow (20 dynes/cm 2 ). b, Quantification of NO production in live cells by DAF-FM imaging after 2 h of treatment. Shown are representative images and mean intensities ± SD from n = 3 biological replicates and statistics calculated using two-tailed, unpaired t test; ** P = 0.0023. c, After 2 h treatment, control and GSK205 treated monolayers were incubated with CellROX probe and imaged to quantify reactive oxygen species. Shown are representative images and mean intensities ± SD from n = 6 biological replicates and statistics calculated using two-tailed, unpaired t test; **** P < 0.0001. d, Control and GSK205 treated monolayers were fixed after 4 h and stained for ICAM-1 and E-Selectin. Shown are representative images and mean intensities ± SD from n = 5 biological replicates and statistics calculated using two-tailed, unpaired t test; **** P < 0.0001 for both graphs. e, Control and GSK205 treated monolayers were fixed after 2 h and stained for DAPI, junctional marker ZO-1 and the p65 subunit of NF-κB. Nuclear expression of NF-κB p65 was quantified by mean fluorescence intensity after nuclear mask application. Shown is mean intensity from n = 115 cells analyzed for each condition for n = 3 biological replicates and statistics calculated using two-tailed, unpaired t test; **** P < 0.0001. f, Experimental design for static TNFα treatment (10 ng/mL; 30 min) in the presence of GSK1016790A (GSK101, 10 nM) or DMSO. g, Following the treatment, monolayers were fixed and stained for DAPI, junctional marker VE-Cadherin and NF-κB p65. Graph represents mean fluorescence intensity in the nucleus from n = 79 cells per group and statistics calculated using one-way ANOVA with post hoc Tukey’s multiple comparisons test; **** P < 0.0001. h, Gene expression was measured by qPCR for TNFα treated monolayers in the presence of DMSO or GSK1016790A (GSK101). Gene expression of NFKBIA, VCAM1, CCL2, SELE and TNF is plotted as a heat map of the mean expression (n = 3 biological replicates). Note that CD31 and ITGB1 are not changed with GSK101 treatment. i, as in (h) with mRNA expression plotted as mean ± SEM for the inflammatory genes, CD31 and ITGB1. * P < 0.05, ** P < 0.01 by two-tailed, unpaired t test. j, Graphical model describing how laminar flow supports localized Trpv4 activation by polarized Caveolin-1 domains, which leads to Ca 2+ activity, eNOS activation, NO production and inhibition of NF-κB mediated transcription.

Article Snippet: Primary antibodies used: Erg (Abcam #ab92513), Ve-cadherin (BD Pharmingen™ #550547), Caveolin-1 (Invitrogen #PA1-064), Caveolin-1 (R&D AF5736), Caveolin-1 (Santa Cruz #sc-894), Cavin-1 (Abcam #ab48824), Icam-1 (Santa Cruz #sc-107), E-selectin (Invitrogen #MA1-06506), ZO-1 (Invitrogen #MA3-39100-A488), NFkB p65 (Cell Signaling #8242S), p-eNOS Ser1177 (Invitrogen #PA5-35879), p-eNOS Ser1177 (Santa Cruz #sc-81510), Trpv4 (Alomone Labs #ACC-034 or LSBio #LS-C401108), VE-Cadherin (R&D #AF938).

Techniques: Imaging, Two Tailed Test, Incubation, Staining, Marker, Expressing, Fluorescence, Activation Assay, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

doi: 10.1101/2023.05.26.542500

Figure Lengend Snippet: a, Flow-aligned HAEC monolayers were treated with DMSO or GSK10116790A for 2 h followed by live cell imaging of DAF-FM and Hoechst. NO was quantified as the DAF-FM mean intensity signal ± SD (n = 4 biological replicates), and statistics were calculated using two-tailed, unpaired t test; * P = 0.0172. For (b-c), confluent monolayers of HAECs were exposed to laminar shear stress (20 dynes/cm 2 ) for 48 h then treated for 30 min. After 30 min, monolayers were fixed and stained for Caveolin-1, ZO-1 and DAPI. Full- length cells were segmented into 3 equal-length regions, and Caveolin-1 staining intensity was quantified for each subcellular region. b, Representative image for flow-aligned HAECs treated with the Trpv4 antagonist GSK205 (20 µM) in the presence of laminar flow (20 dynes/cm 2 ) for 30 min. Caveolin-1 staining intensity is quantified for 67 cells. Bar graph displays the mean ± SD with data analyzed by one-way ANOVA and post hoc Tukey’s multiple comparisons test. **** P < 0.0001 (Upstream vs. Downstream; Mid vs. Downstream). c, Representative image for flow-aligned HAECs removed from flow for 30 min then fixed and stained. Caveolin-1 staining intensity is quantified for 61 cells. Bar graph displays the mean ± SD with data analyzed by one-way ANOVA and post hoc Tukey’s multiple comparisons test. **** P < 0.0001 (Upstream vs. Downstream; Mid vs. Downstream). d, Flow-aligned HAEC monolayers were treated for 30 min in static conditions with DMSO or TNFα (10 ng/mL) in the presence or absence of GSK1016790A (10 nM). After 30 min, monolayers were fixed and stained for junctional marker ZO-1, inflammatory marker ICAM-1 and DAPI. ICAM-1 mean fluorescence intensity ± SD (n = 3 biological replicates with 2 images quantified per slide) did not show statistical difference across the groups. ns, not significant by two-tailed, unpaired t test. e, To measure reactive oxygen species (ROS), cells were loaded with CM-H 2 DCFDA (5 μM, DCF), then flow aligned for 48 h, followed by 1 h treatment in static conditions with DMSO, 10 ng/mL TNFα + DMSO or 10 ng/mL TNFα + 10 nM GSK1016790A. Mean DCF intensity signal ± SD is shown from 60 cells/condition. Data analyzed by one-way ANOVA and post hoc Tukey’s multiple comparisons test; **** P < 0.0001 or not significant (ns).

Article Snippet: Primary antibodies used: Erg (Abcam #ab92513), Ve-cadherin (BD Pharmingen™ #550547), Caveolin-1 (Invitrogen #PA1-064), Caveolin-1 (R&D AF5736), Caveolin-1 (Santa Cruz #sc-894), Cavin-1 (Abcam #ab48824), Icam-1 (Santa Cruz #sc-107), E-selectin (Invitrogen #MA1-06506), ZO-1 (Invitrogen #MA3-39100-A488), NFkB p65 (Cell Signaling #8242S), p-eNOS Ser1177 (Invitrogen #PA5-35879), p-eNOS Ser1177 (Santa Cruz #sc-81510), Trpv4 (Alomone Labs #ACC-034 or LSBio #LS-C401108), VE-Cadherin (R&D #AF938).

Techniques: Live Cell Imaging, Two Tailed Test, Staining, Marker, Fluorescence