ws12  (Alomone Labs)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Nicorandil Attenuates LPS-Induced Acute Lung Injury by Pulmonary Endothelial Cell Protection via NF-κB and MAPK Pathways

doi: 10.1155/2019/4957646

Figure Lengend Snippet: Nico ameliorated LPS-induced ALI and inflammation. (a) Nico increased LPS-induced Kir6.1 and Kir6.2 downregulation in the lung. (b, c) Lung sections stained with H E showed severe injury in the LPS group which was attenuated by Nico pretreatment. The data revealed a high score for the LPS-treated group which was decreased in the Nico-pretreated group. (d) Nico pretreatment significantly reduced LPS-induced protein leakage in BALF. (e, f) Nico alleviated LPS-induced increments of MPO activities in BALF and lung homogenate. (g, h) Nico prevented the production of TNF- α and IL-1 β in lung homogenate. Data were shown as mean ± SEM ( n = 6 − 8). Statistically significant differences: ∗ P

Article Snippet: Then, the transferred membranes were incubated with primary antibodies against Kir6.1 (Alomone Labs, Jerusalem, Israel), Kir6.2 (Abcam), NF-κ B p-p65/p65, p-iκ B-α /iκ B-α , p-p38/p38, p-ERK/ERK, p-JNK/JNK, intercellular adhesion molecule-1 (ICAM-1), cleaved-caspase-3 (c-caspase-3), caspase-9 (1 : 1000, Cell Signaling Technology), endothelial nitric oxide synthase (eNOS) (1 : 1000, Santa Cruz), inducible nitric oxide synthase (iNOS) (1 : 1000, Millipore), CCAAT/enhancer-binding protein homologous protein (CHOP), vascular cell adhesion molecule-1 (VCAM-1), VE-cadherin, Nox4 (1 : 1000), MnSOD (1 : 5000, Abcam), and β -actin (1 : 5000, Proteintech, Rosemont, USA) overnight.

Techniques: Staining

Journal: PLoS ONE

Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

doi: 10.1371/journal.pone.0104686

Figure Lengend Snippet: Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P

Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

Techniques: Isolation, Mass Spectrometry, Activity Assay

Journal: PLoS ONE

Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

doi: 10.1371/journal.pone.0104686

Figure Lengend Snippet: Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels. A : (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B : Representative force myograph trace obtained from an ovx artery. C and D : Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their ( C ) change in tone and ( D ) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C : Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D : Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI 2 ) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

doi: 10.1371/journal.pone.0104686

Figure Lengend Snippet: IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels. A : Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B : Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P

Article Snippet: Further, blocking IK1 channels alone with 1 µM tram34 had little effect on control and ovx arteries (control: 2.7±0.9; ovx: 8.0±1.8; n = 8; P > 0.05; ).

Techniques: Activity Assay, Mouse Assay

Journal: bioRxiv

Article Title: Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1101/597088

Figure Lengend Snippet: Na v 1.5 and V m regulate Rac1-GTP colocalization with phosphatidylserine. (A) Images of representative cells after treatment with TTX (30 µM) and NS-1619 (1 µM) for 3 h. Cells were labeled with Rac1-GTP antibody (green), annexin V (red) and DAPI (blue). Dashed lines highlight regions of interest at the leading edge. (B) Cytofluorogram showing colocalization of annexin V and Rac1-GTP staining in region of interest in control cell from (A), normalized to maximum in each channel. (C) Cytofluorogram showing colocalization of annexin V and Rac1-GTP staining in region of interest in TTX cell from (A), normalized to maximum in each channel. (D) Cytofluorogram showing colocalization of annexin V and Rac1-GTP staining in region of interest in NS-1619 cell from (A), normalized to maximum in each channel. (E) Manders’ corrected colocalization coefficients for annexin V and Rac1-GTP staining in regions of interest of cells after treatment with TTX (30 µM) and NS-1619 (1 µM) for 3 h (n = 30). (F) Li’s intensity correlation quotient for Rac1-GTP and annexin V colocalization (n = 30). Data are mean and SEM. **P

Article Snippet: ChemicalsIberiotoxin, NS-1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1101/597088

Figure Lengend Snippet: Na v 1.5 and V m regulate Rac1 activation/distribution. (A) Images of representative cells after treatment with TTX (30 µM) and NS-1619 (1 µM) for 3 h. Cells were labeled with Rac1-GTP antibody (green), phalloidin (red) and DAPI (blue). Arrows in the Rac1-GTP panels highlight the distribution or lack of expression at the leading edge. (B) Rac1-GTP signal density, measured across 20 arcs, in 0.43 µm radius increments, within a quadrant mask region of interest at the leading edge, normalized to the first arc (n ≥ 66). (C) Peak Rac1-GTP signal density per cell from (B), normalized to the first arc (n ≥ 66). (D) Total Rac1-GTP quantified in whole cell lysates using colorimetric small GTPase activation assay (n = 6). (E) Images of representative cells after treatment with TTX (30 µM) and NS-1619 (1 µM) for 3 h. Cells were labeled with Rac1-GTP antibody (green), total Rac1 antibody (red), and DAPI (blue). Arrows in the Rac1-GTP panels highlight the distribution or lack of expression at the leading edge. (F) Total Rac1 signal density, measured across 20 arcs, in 0.43 µm radius increments, within a quadrant mask region of interest at the leading edge, normalized to the first arc (n ≥ 59). (G) Peak Rac1 signal density per cell from (F), normalized to the first arc (n ≥ 59). (H) Ratio of Peak Rac1-GTP/Peak total Rac1 for each experimental repeat, normalized to control (n = 3). Data are mean and SEM. *P

Article Snippet: ChemicalsIberiotoxin, NS-1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Activation Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1101/597088

Figure Lengend Snippet: Na v 1.5-dependent membrane potential depolarization regulates cell migration. (A) Representative scratch wounds at 0 h and 6 h into a wound healing assay ± TTX (30 μM) or NS-1619 (1 μM). Red dotted lines highlight wound edges. (B) Wound area during the migration assay (“gap remaining”), normalized to starting value (n = 3). (C) t 1/2 of wound closure (n ≥ 5). (D) Collective migration (µm/h) of cells closing the wound (n ≥ 5). (E) Instantaneous velocity (µm/s) of segmented cells (n ≥ 2662). (F) Polar histograms showing directionality of migrating cells at the leading edge of wounds (P

Article Snippet: ChemicalsIberiotoxin, NS-1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Migration, Wound Healing Assay

Journal: bioRxiv

Article Title: Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1101/597088

Figure Lengend Snippet: The large conductance Ca 2+ -activated K + channel K Ca 1.1 regulates the membrane potential but not intracellular Na + . (A) MDA-MB-231 cells labeled with K Ca 1.1 antibody (green), phalloidin to label the actin cytoskeleton (red), and DAPI to label the nucleus (blue). Western blot of K Ca 1.1 in control MDA-MB-231 cells and cells in which Na v 1.5 has been knocked down with shRNA. Positive control = rat brain lysate. Loading control = α -tubulin. Representative perforated patch clamp recording showing activation of outward current using the K Ca 1.1 activator (NS-1619; 1 µM) and inhibition with iberiotoxin (100 nM). The cell was held at −120 mV for 250 ms before depolarization to +60 mV for 300 ms. (D) Current-voltage relationship of the K Ca 1.1 current. Cells were held at −120 mV for 250 ms before depolarization to voltages ranging from −60 to +90 mV in 10 mV steps for 300 ms (n = 5). Data are fitted with single exponential functions. (E) Dose-dependent effect of NS-1619 on the steady-state V m (n ≥ 6). Data are fitted to a sigmoidal logistic function. (F) Effect of NS-1619 (1 µM) on steady-state V m (n = 12). (G) Effect of NS-1619 (1 µM, 5 min) on [Na + ] i (n = 22). (H) V m recorded using intracellular solution with free [Ca 2+ ] buffered to 5.7 nM vs. 100 nM (n ≥ 10). Data are mean and SEM. **P

Article Snippet: ChemicalsIberiotoxin, NS-1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Multiple Displacement Amplification, Labeling, Western Blot, shRNA, Positive Control, Patch Clamp, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1101/597088

Figure Lengend Snippet: Na v 1.5-dependent membrane potential depolarization regulates lamellipodia formation. (A) Images of representative cells after treatment with TTX (30 μM) or NS-1619 (1 μM) for 3 h. Cells were fixed and stained with phalloidin (red) and DAPI (blue). Lower row shows masks of cells in the upper row, from which the circularity was calculated. (B) Circularity (n ≥ 61). (C) Feret’s diameter (µm; n ≥ 57). (D) Number of MDA-MB-231 cells with a lamellipodium (P

Article Snippet: ChemicalsIberiotoxin, NS-1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Staining, Multiple Displacement Amplification

Journal: Journal of Cellular Physiology

Article Title: Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration, et al. Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1002/jcp.29290

Figure Lengend Snippet: Na v 1.5‐dependent membrane potential depolarization regulates lamellipodia formation. (a) Images of representative cells after treatment with TTX (30 μM) or NS‐1619 (1 μM) for 3 hr. Cells were fixed and stained with phalloidin (red) and DAPI (blue). Lower row shows masks of cells in the upper row, from which the circularity was calculated. (b) Circularity ( n ≥ 61). (c) Feret's diameter (µm; n ≥ 57). (d) Number of MDA‐MB‐231 cells with a lamellipodium ( p

Article Snippet: 2.1 ChemicalsIberiotoxin, NS‐1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Staining, Multiple Displacement Amplification

Journal: Journal of Cellular Physiology

Article Title: Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration, et al. Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1002/jcp.29290

Figure Lengend Snippet: Na v 1.5 and V m regulate Rac1‐GTP colocalization with phosphatidylserine. (a) Images of representative cells after treatment with TTX (30 µM) and NS‐1619 (1 µM) for 3 hr. Cells were labeled with Rac1‐GTP antibody (green), annexin V (red), and DAPI (blue). Dashed lines highlight regions of interest at the leading edge. (b) Cytofluorogram showing colocalization of annexin V and Rac1‐GTP staining in region of interest in control cell from (a), normalized to maximum in each channel. (c) Cytofluorogram showing colocalization of annexin V and Rac1‐GTP staining in region of interest in TTX cell from (a), normalized to maximum in each channel. (d) Cytofluorogram showing colocalization of annexin V and Rac1‐GTP staining in region of interest in NS‐1619 cell from (a), normalized to maximum in each channel. (e) Manders' corrected colocalization coefficients for annexin V and Rac1‐GTP staining in regions of interest of cells after treatment with TTX (30 µM) and NS‐1619 (1 µM) for 3 hr ( n = 30). (f) Li's intensity correlation quotient for Rac1‐GTP and annexin V colocalization ( n = 30). Data are mean and SEM . ** p

Article Snippet: 2.1 ChemicalsIberiotoxin, NS‐1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Labeling, Staining

Journal: Journal of Cellular Physiology

Article Title: Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration, et al. Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1002/jcp.29290

Figure Lengend Snippet: The large conductance Ca 2+ ‐activated K + channel K Ca 1.1 regulates the membrane potential but not intracellular Na + . (a) MDA‐MB‐231 cells labeled with K Ca 1.1 antibody (green), phalloidin to label the actin cytoskeleton (red), and DAPI to label the nucleus (blue). (b) Western blot of K Ca 1.1 in control MDA‐MB‐231 cells and cells in which Na v 1.5 has been knocked down with shRNA. Positive control = rat brain lysate. Loading control = α‐tubulin. (c) Representative perforated patch clamp recording showing activation of outward current using the K Ca 1.1 activator (NS‐1619; 1 µM) and inhibition with iberiotoxin (100 nM). The cell was held at −120 mV for 250 ms before depolarization to + 60 mV for 300 ms. (d) Current–voltage relationship of the K Ca 1.1 current. Cells were held at −120 mV for 250 ms before depolarization to voltages ranging from −60 to +90 mV in 10 mV steps for 300 ms ( n = 5). Data are fitted with single exponential functions. (e) Dose‐dependent effect of NS‐1619 on the steady‐state V m ( n ≥ 6). Data are fitted to a sigmoidal logistic function. (f) Effect of NS‐1619 (1 µM) on steady‐state V m ( n = 12). (g) Effect of NS‐1619 (1 µM, 5 min) on [Na + ] i ( n = 22). (h) V m recorded using intracellular solution with free [Ca 2+ ] buffered to 5.7 nM versus 100 nM ( n ≥ 10). Data are mean and SEM. ** p

Article Snippet: 2.1 ChemicalsIberiotoxin, NS‐1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Multiple Displacement Amplification, Labeling, Western Blot, shRNA, Positive Control, Patch Clamp, Activation Assay, Inhibition

Journal: Journal of Cellular Physiology

Article Title: Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration, et al. Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1002/jcp.29290

Figure Lengend Snippet: Na v 1.5 and V m regulate Rac1 activation/distribution. (a) Images of representative cells after treatment with TTX (30 µM) and NS‐1619 (1 µM) for 3 hr. Cells were labeled with Rac1‐GTP antibody (green), phalloidin (red), and DAPI (blue). Arrows in the Rac1‐GTP panels highlight the distribution or lack of expression at the leading edge. (b) Rac1‐GTP signal density, measured across 20 arcs, in 0.43 µm radius increments, within a quadrant mask region of interest at the leading edge, normalized to the first arc ( n ≥ 66). (c) Peak Rac1‐GTP signal density per cell from (b), normalized to the first arc ( n ≥ 66). (d) Total Rac1‐GTP quantified in whole‐cell lysates using colorimetric small GTPase activation assay ( n = 6). (e) Images of representative cells after treatment with TTX (30 µM) and NS‐1619 (1 µM) for 3 hr. Cells were labeled with Rac1‐GTP antibody (green), total Rac1 antibody (red), and DAPI (blue). Arrows in the Rac1‐GTP panels highlight the distribution or lack of expression at the leading edge. (f) Total Rac1 signal density, measured across 20 arcs, in 0.43 µm radius increments, within a quadrant mask region of interest at the leading edge, normalized to the first arc ( n ≥ 59). (g) Peak Rac1 signal density per cell from (f), normalized to the first arc ( n ≥ 59). (h) Ratio of Peak Rac1‐GTP/Peak total Rac1 for each experimental repeat, normalized to control ( n = 3). Data are mean and SEM . * p

Article Snippet: 2.1 ChemicalsIberiotoxin, NS‐1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Activation Assay, Labeling, Expressing

Journal: Journal of Cellular Physiology

Article Title: Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration, et al. Voltage‐dependent activation of Rac1 by Nav1.5 channels promotes cell migration

doi: 10.1002/jcp.29290

Figure Lengend Snippet: Na v 1.5‐dependent membrane potential depolarization regulates cell migration. (a) Representative scratch wounds at 0 hr and 6 hr into a wound healing assay ± TTX (30 μM) or NS‐1619 (1 μM). Red dotted lines highlight wound edges. (b) Wound area during the migration assay (“gap remaining”), normalized to starting value ( n = 3). (c) t 1/2 of wound closure ( n ≥ 5). (d) Collective migration (µm/hr) of cells closing the wound ( n ≥ 5). (e) Instantaneous velocity (µm/s) of segmented cells ( n ≥ 2,662). (f) Polar histograms showing directionality of migrating cells at the leading edge of wounds ( p

Article Snippet: 2.1 ChemicalsIberiotoxin, NS‐1619, tetrodotoxin (TTX) and veratridine were purchased from Alomone Labs.

Techniques: Migration, Wound Healing Assay