mittx  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2 expression in spinal cord samples from WT and EAE mice. ( A ) Immunohistochemical staining of TRPV2 in spinal cord of control and EAE-induced mice in the onset, peak and in the chronic phase, during remission. In control conditions, TRPV2 is expressed in neurons in GM (inset, arrows), and in oligodendrocytes in WM (inset, arrowheads). After EAE induction, an increase in TRPV2 expression is mainly located in inflammatory focuses at the peak clinical symptomatology in WM (empty arrowheads). ( B ) MSRA and TRPV2 protein expression in thoracic spinal cord samples from CFA and MOG mice (9, 14, 21 and 28 days post-immunization). ( B ) A significant increase in TRPV2 protein levels (#, p = 0.0014) is determined in MOG14 mice when compared with CFA ( p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2-centered cellular cross-talk model in physiological and pathophysiological myelination. During embryonic development, TRPV2 develops an essential role in neurite and axon outgrowth. In that period, TRPV2 is known to be expressed and activated in neurons, and putatively in OPCs, where possible cross-talk between both cells might be triggered through TRPV2 activation. In adulthood, TRPV2 expression is found in microglia, neurons and oligodendrocytes. Upon an insult such as demyelination, TRPV2, as a non-selective ion channel, is activated. Some known triggering stimuli are NO, LPC or oxidative stress, the latter causing methionine oxidation which facilitates TRPV2 sensitization. TRPV2 activation promotes a sodium/calcium inward influx from extracellular media and/or ER or endosomes. Consequently, cells are depolarized, which initiate processes such as actin polymerization or phagocytosis in microglia or innate immune cells. In those cells, TRPV2 has been involved in migration, phagocytosis or cytokine production. TRPV2 inactivation is promoted by MSRA enzyme, by reducing oxidized methionine. In our results, we observed both an increase of TRPV2 and MSRA in demyelinating scenarios. During remyelination, OPCs proliferate and migrate to the demyelinated area, where they differentiate through several stages to mature oligodendrocytes, and start myelination. Our results showed increased TRPV2 expression during this period. Overall, ubiquitous TRPV2 expression and activation in de- and remyelinating processes suggest a multicellular cross-talk that promotes myelin repair at all levels. TRPV2 is relevant in development and demyelination, making this model suitable for the recapitulation hypotheses. Created with BioRender.com (accessed on 10 January 2022) [ 8 , 9 , 10 ].

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Activation Assay, Expressing, Migration

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2 expression in mixed glia cultures of mice after pro-inflammatory (LPS), anti-inflammatory (IL-4) and demyelinating (LPC) treatments. ( A – I ) Double immunohistochemical staining allowed for the determination of whether TRPV2 was expressed in microglia ( A – C ), astrocytes ( D – F ) or oligodendrocyte cells ( G – I ). TRPV2 was highly expressed in the cell body of some microglia (full arrowheads) but not all of them (empty arrowheads) in control conditions, and TRPV2 was spread afterwards through the cell body of activated microglia cells, with an expression pattern highly coincident with CD68+ lysosomes (arrows). While no expression of TRPV2 was found in astrocytes (empty arrowheads), OPCs showed either low or absent expression in control conditions (full and empty arrowheads, respectively) and increased expression throughout the cell body (arrows) and the principal soma (arrowheads) after both pro-inflammatory and anti-inflammatory treatments. ( J , K ) Quantification of TRPV2 protein (ng/mL) by ELISA in secondary mixed glial cultures in control conditions, and after LPS and IL-4 treatments ( J ) and in quaternary mixed glial cultures, containing myelin-binding protein (MBP)+ oligodendrocytes, in demyelinating conditions ( K ). Determination of TRPV2 showed a significant decrease in TRPV2 after LPS and LPC treatments ( J , K ), and an increase after IL-4 treatment ( p = 0.05) compared to control conditions ( J ). ( L , M ) Quantification of NO secondary mixed glial cultures in control conditions, and after LPS and IL-4 treatments ( L ) and in quaternary mixed glial cultures in demyelinating conditions ( M ). An increase in NO concentration was observed in cell cultures after LPS treatment compared to control conditions, while a significant decrease was observed after IL-4 treatment. After LPC treatment, NO remained stable compared to the control. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison compared to the control in the LPS and IL-4 treated cultures (* p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2, Opalin and MSRA expression in spinal cord of wild-type (WT) and hypomyelination jimpy mice. Immunohistochemical staining against MBP ( A , B ), TRPV2 ( D , E ), Opalin ( G , H ) and MSRA ( J , K ) were performed in spinal cord sections of 21-day-old WT and jimpy mice, and immunoreactivity was quantified and analyzed as Area x Intensity. Results show that MBP was importantly reduced in both GM ( a , b ) and WM ( a’ , b’ ) of jimpy mice compared to WT ( A – C ). TRPV2 expression was mainly located in neurons in GM ( d’ ) and glial cells, putatively oligodendrocytes, and in WM ( d ) of WT. A significant decrease in TRPV2 expression in the spinal cord of jimpy mice was observed compared to WT ( E , F , e , e’ ). Opalin expression was importantly found in WM of WT mice ( G , g ), and to a lesser extent also in GM ( g’ ), but showed a marked decrease in WM and GM of jimpy mice ( H – I , h , h’ ). MSRA was the only molecule that did not suffer an important reduction of its expression in both WM ( j , k ) and GM ( j’ , k’ ) in jimpy mice ( L ). Results were expressed as mean ± SEM. Statistical analysis of the results were performed with an unpaired Student’s t -test (** p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2 and MSRA expression in WM of cuprizone-induced demyelination and remyelination in mice. ( A – I ) Representative images of immunohistochemical staining directed against MBP, TRPV2 and MSRA in the CC of control mice ( A , D , G ) and cuprizone-intoxicated mice after demyelination ( B , E , H , 5 weeks of treatment) and during remyelination ( C , F , I , 5 weeks of treatment + 1 week of normal diet). ( A – C ) MBP immunostaining shows the myelinated CC of WT mice in basal conditions ( A ). At 5 weeks of cuprizone treatment, the CC is largely demyelinated ( B ) with a myelination-resistant area (*), and after one week of normal diet, the CC is mostly remyelinated ( C ). Control mice show undetectable levels of TRPV2 and MSRA in the CC ( D and G ). After demyelination, variably low and high levels of TRPV2 were observed in cell bodies (empty arrowheads and arrowheads, respectively in E , F ). High levels of TRPV2 were clearly observed at remyelination (inset in F ). During remyelination, most TRPV2+ cells co-expressed APC (full arrowheads in L ), a marker for mature oligodendrocytes. In the CC area resistant to demyelination (* in E , F ), TRPV2+ cells showed high levels of expression in both conditions. An increase in MSRA levels of expression were also observed in cell bodies after demyelination and remyelination (arrowheads in H and I ). In demyelination, most cells expressed low levels of MSRA in the CC (inset in H ), while during remyelination, a lower number of MSRA+ cells expressing higher levels of MSRA were observed (inset in I ). ( J – K ) Quantification of TRPV2 and MSRA expression after cuprizone treatment. ( J ) Quantitative analysis of TRPV2 immunoreactivity in the CC confirmed the progressive increase of TRPV2 expression in demyelinating and remyelinating conditions compared to control mice, which was statistically significant in the latter. ( K ) Quantitative analysis of MSRA immunoreactivity in the CC showed that the increase of MSRA expression during demyelination was statistically significant when compared to control mice. Data are shown as ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test (** p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining, Immunostaining, Marker

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: MSRA and TRPV2 expression in frontal cortex samples from healthy subjects ( n = 4) and MS ( n = 6) patients. ( A ) TRPV2 mRNA ( p = 0.3314) is not changed between MS and healthy samples. ( B ) A significant upregulation in MSRA mRNA ( p = 0.0014) is found in MS; ( C ) A significant decrease in TRPV2 protein levels ( p = 0.0232) and a significant increase in MSRA protein levels ( p = 0.0124) were determined in MS samples by western blot. Bars show mean ± SEM; * p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: TRPV2: A Key Player in Myelination Disorders of the Central Nervous System

doi: 10.3390/ijms23073617

Figure Lengend Snippet: TRPV2 interaction with Opalin, NTM and PLP1 in vitro during basal and inflammatory conditions. ( A ) Rat TRPV2 FL protein and rat ARD of TRPV2 were immobilized on membranes and incubated with increasing concentrations of rat protein brain lysates (0–10 µg). Opalin, NTM and PLP1 were detected by immunoblot. ( B ) Immunocytochemistry in mouse mixed glial primary cultures shows TRPV2 (green), Opalin (red) and DAPI (blue). TRPV2-Opalin colocalization is shown as yellow signal (merge) and both proteins are co-expressed in some cells (arrowheads). Expression of these proteins is mainly found in the membrane. ( C ) TRPV2–Opalin colocalization expressed as Pearson’s coefficient in basal and inflammatory conditions. Both proteins show a high degree of colocalization. ( D ) TRPV2 colocalization with Opalin, expressed as Mander’s coefficient M2 (fraction of TRPV2 signal overlapping Opalin) in basal and inflammatory conditions. Both proteins show a high degree of colocalization. Results were expressed as mean ± SEM. Statistical analysis of the results were performed with an unpaired Student’s t -test ( p

Article Snippet: After washes with 0.1 M TBS, sections were incubated for 1 h with BB, and then, overnight with rabbit anti-TRPV2 (1:100, Alomone) diluted in BB at 4 °C and 1 h at RT ( ).

Techniques: In Vitro, Incubation, Immunocytochemistry, Expressing

Journal: Science signaling

Article Title: Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

doi: 10.1126/scisignal.aan2694

Figure Lengend Snippet: Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.

Article Snippet: Cells were fixed with 3.2% formaldehyde/0.1% glutaraldehyde–phosphate-buffered saline (PBS), permeabilized and blocked with 0.2% saponin/5% horse serum–PBS, and incubated with primary antibodies against α-tubulin (1:100; MA1-80017, Life Technologies), BKα (1:100; APC-021, Alomone Labs), and RyR2 (1:100; ab2868, Abcam).

Techniques: Isolation, Immunolabeling

Journal: PLoS ONE

Article Title: Hemodynamic Forces Regulate Developmental Patterning of Atrial Conduction

doi: 10.1371/journal.pone.0115207

Figure Lengend Snippet: Stretching induces conduction marker expression in the atria. Whole embryonic hearts were isolated at HH Stage 18 and placed in culture for 8 hrs. A ) Image of control heart (left lateral view) following 8 hrs of culture with vehicle (Tyrode's solution). B ) Image of heart in which a bead of silicone oil was microinjected into the atria following 8 hrs. of culture with vehicle. C ) Image of silicone injected atria cultured for 8 hrs with 5 uM GsMTx4. D ) Quantification of gene expression levels in atria following whole heart culture. P-values indicated significance relative to control (***p

Article Snippet: To block stretch activated responses hearts were cultured with 5 uM GsMTx4 (Alomone Labs, STG-100) dissolved in Tyrode's solution (pH 7.4).

Techniques: Marker, Expressing, Isolation, Injection, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Fluid shear stress-dependent Ca 2+ influx in Meg-01 cells is inhibited by GsMTx-4 and chelation of extracellular Ca 2+ . A and B , representative images ( A ) and F / F 0 fluorescence recordings ( B ) of single Meg-01 cells exposed to arterial and two levels of stenotic shear in HBSS with Ca 2+ , without Ca 2+ (EGTA), and with GsMTx-4 in the presence of Ca 2+ . C , mean peak F / F 0 increases ( n = 53 cells) in response to different shear levels in the presence of extracellular Ca 2+ . D , mean peak F / F 0 increases under no flow conditions ( n = 113 cells) and at the high stenotic shear rate with ( n = 85 cells) and without extracellular Ca 2+ ( n = 35 cells) and with GsMTx-4 in the presence of Ca 2+ ( n = 37 cells). ****, p

Article Snippet: Materials The MS ion channel inhibitor GsMTx-4 peptide (STG-100) was from Alomone Labs (Jerusalem, Israel), and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience (Bristol, UK).

Techniques: Fluorescence, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: GsMTx-4 inhibits thrombus formation independently of P2X1 receptors. A , P2X1-dependent Ca 2+ entry (α,β-meATP, 10 μ m ) in platelet suspensions is completely inhibited by 1 μ m NF449. B , effect of 2.5 μ m GsMTx-4 and 1 μ m NF449, individually and combined, on thrombi formed on a collagen surface. The average values are shown ( n = 7) for thrombus volume ( panel i ), surface coverage ( panel ii ), and thrombus height ( panel iii ). *, p

Article Snippet: Materials The MS ion channel inhibitor GsMTx-4 peptide (STG-100) was from Alomone Labs (Jerusalem, Israel), and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Effect of GsMTx-4 on Ca 2+ entry via TRPC6, P2X1, and store-operated channels in platelets. A–C , representative [Ca 2+ ] i recordings ( left panels ) and average peak [Ca 2+ ] i responses ( right panels ) for store-operated ( n = 4) ( A ), TRPC6 ( n = 4) ( B ), and P2X1 cation channels ( n = 3) ( C ) in suspensions of platelets in the presence and absence of GsMTx-4. Store-operated Ca 2+ entry was assessed by addition of 1.26 m m CaCl 2 15 min after treatment with the SERCA inhibitor thapsigargin. TRPC6 was activated using the diacylglycerol analogue OAG. P2X1 was activated with the non-hydrolyzable ATP analogue α,β-meATP (10 μ m ). **, p

Article Snippet: Materials The MS ion channel inhibitor GsMTx-4 peptide (STG-100) was from Alomone Labs (Jerusalem, Israel), and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Collagen-induced thrombus formation but not platelet aggregation is inhibited by GsMTx-4. A , representative images of surface coverage and 3D Z -stacks for thrombi formed by DiOC 6 -stained platelets on a collagen surface under control and GsMTx-4-pretreated conditions. Scale bars , 20 μm. B.F. , bright field. B , average values ( n = 6) for thrombus height, thrombus volume, and surface coverage under control and GsMTx-4-treated conditions. C , collagen-evoked aggregation under control and GsMTx-4-treated conditions. Integrilin treatment was performed as a control to demonstrate that aggregation is abolished by inhibition of the α IIb β 3 integrin. Representative light transmission traces are shown in the left panel , and average maximal light transmission responses expressed as percentages of aggregation are shown in the right panel ( n = 3). **, p

Article Snippet: Materials The MS ion channel inhibitor GsMTx-4 peptide (STG-100) was from Alomone Labs (Jerusalem, Israel), and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience (Bristol, UK).

Techniques: Staining, Inhibition, Transmission Assay

Journal: The Journal of Biological Chemistry

Article Title: Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

doi: 10.1074/jbc.M116.766196

Figure Lengend Snippet: Fluid shear stress induces Ca 2+ transients in single platelets that are inhibited by GsMTx-4 and chelation of extracellular Ca 2+ . A , a cartoon representation of single platelet attachment to PECAM-1 antibody-coated biochip surface via the Ig domains 1 and 2 of the platelet PECAM-1. B , representative Fluo-3 fluorescence and bright field ( B.F. ) images of individual Fluo-3-loaded attached platelets before and during exposure to arterial shear. Scale bars , 20 μm. The magnified rectangular sections have been enlarged 3-fold. C , representative F / F 0 Fluo-3 recordings in single platelets during no applied shear stress ( white regions ) and normal arterial shear ( gray regions ). Two successive cycles of 4 min without shear followed by 4 min of arterial shear were applied, in which the second cycle was used to compare the control conditions ( i.e. HBSS + Ca 2+ only) ( panel i ), with the effect of GsMTx-4 ( panel ii ), or removal of extracellular Ca 2+ ( panel iii ). D–G , average Ca 2+ increases, calculated as the 4-min F / F 0 integral of all [Ca 2+ ] i transients. D , responses in the absence of shear and during arterial shear, in the presence of extracellular Ca 2+ with and without GsMTx-4 and in the absence of extracellular Ca 2+ ( n = 46, 46, 23, and 14 cells in no shear, HBSS, GsMTx-4, and EGTA, respectively). *, p

Article Snippet: Materials The MS ion channel inhibitor GsMTx-4 peptide (STG-100) was from Alomone Labs (Jerusalem, Israel), and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience (Bristol, UK).

Techniques: Fluorescence