egta  (Tocris)

 
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    Name:
    EGTA
    Description:
    Calcium chelating agent
    Catalog Number:
    2807
    Price:
    None
    Category:
    Biochemicals and Molecular Biology Reagents
    Formula:
    Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
    Buy from Supplier


    Structured Review

    Tocris egta
    EGTA
    Calcium chelating agent
    https://www.bioz.com/result/egta/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila"

    Article Title: Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila

    Journal: International Journal of Peptides

    doi: 10.1155/2013/573716

    Nociceptin-NH2 is a depolarizing signal in Tetrahymena thermophila . (a) 5 μ M nociceptin-NH2 causes a depolarization of approximately 20 mV, though this concentration does not often provoke a behavioral response in Tetrahymena. (b) 50 μ M nociceptin-NH2 causes a depolarization of approximately 40 mV. This concentration is the EC 100 for behavioral avoidance in Tetrahymena. (c) The depolarization produced by 50 μ M nociceptin-NH2 is eliminated by the addition of 1 mM EGTA to the external medium, implying that calcium is involved in the depolarization.
    Figure Legend Snippet: Nociceptin-NH2 is a depolarizing signal in Tetrahymena thermophila . (a) 5 μ M nociceptin-NH2 causes a depolarization of approximately 20 mV, though this concentration does not often provoke a behavioral response in Tetrahymena. (b) 50 μ M nociceptin-NH2 causes a depolarization of approximately 40 mV. This concentration is the EC 100 for behavioral avoidance in Tetrahymena. (c) The depolarization produced by 50 μ M nociceptin-NH2 is eliminated by the addition of 1 mM EGTA to the external medium, implying that calcium is involved in the depolarization.

    Techniques Used: Concentration Assay, Produced

    Calcium chelators inhibit the behavioral response to 50 μ M nociceptin-NH2 in Tetrahymena thermophila . EGTA (closed circles) reduces avoidance to 20% (near baseline) at a concentration of 50 μ M. The IC 50 of EGTA is approximately 7.5 μ M. Thapsigargin (open triangles) reduced avoidance by 50% at a concentration of 100 μ M; however, increasing the concentration to 300 μ M did not cause a significant decrease in avoidance beyond that seen with 100 μ M thapsigargin. N ≥ 6. N represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance.
    Figure Legend Snippet: Calcium chelators inhibit the behavioral response to 50 μ M nociceptin-NH2 in Tetrahymena thermophila . EGTA (closed circles) reduces avoidance to 20% (near baseline) at a concentration of 50 μ M. The IC 50 of EGTA is approximately 7.5 μ M. Thapsigargin (open triangles) reduced avoidance by 50% at a concentration of 100 μ M; however, increasing the concentration to 300 μ M did not cause a significant decrease in avoidance beyond that seen with 100 μ M thapsigargin. N ≥ 6. N represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance.

    Techniques Used: Concentration Assay

    2) Product Images from "Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity"

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    Journal: bioRxiv

    doi: 10.1101/2019.12.18.881391

    Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.
    Figure Legend Snippet: Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Techniques Used: Infection, Luciferase, Activity Assay, Standard Deviation

    3) Product Images from "Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7"

    Article Title: Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00370

    Pak1 regulates L-selectin shedding in activated CD4 + T cells. (A) Analysis by ELISA of soluble L-selectin in the supernatants of WT and Pak1 (T) −/− T cell blasts. Data were pooled from six independent experiments. (B) Representative time-dependent calcium flux (indo-violet/indo-blue) of Indo-1AM loaded WT and Pak1 (T) −/− CD4 + T cell blasts, stimulated with CCL21 (200 ng/mL) for the indicated time. Results from one of three independent experiments are shown. (C) Calmodulin (CaM) was immunoprecipitated and eluted material was blotted for co-precipitation L-selectin and CaM in lysates prepared from WT and Pak1 (T) −/− T cell blasts. Whole-cell lysates were blotted for CaM. Results from two independent experiments are shown. (D) Analysis of soluble L-selectin in the supernatant of WT and Pak1 (T) −/− CD4 + T cell blasts pre-treated for 20 min with DMSO (1:1,000), BAPTA-AM 10 μM and EGTA 2 mM, or TMI1 (0.5 μM), and incubated for 30 min in the presence of 100 ng/mL CCL21. (E) Left, L-selectin surface marker expression as measured by flow cytometry from WT and Pak1 (T) −/− CD4 + T cell blasts cultured with or without TMI1(10 μM) for 6 days. The results are representative of four experiments. Right, bar chart shows MFI (%) of L-selectin in WT and Pak1 (T) −/− blast T cells. (F) Percentage of WT, Pak1 (T) −/− and Pak1 (T) −/− treated with TMI1 cells per field of view. Statistical analysis: unpaired Student's t -test (A) ; ANOVA between selected columns with Sidak's multiple comparison test (D) ; ANOVA with Tukey's multiple comparison test (E,F) . ns , not significant; * P
    Figure Legend Snippet: Pak1 regulates L-selectin shedding in activated CD4 + T cells. (A) Analysis by ELISA of soluble L-selectin in the supernatants of WT and Pak1 (T) −/− T cell blasts. Data were pooled from six independent experiments. (B) Representative time-dependent calcium flux (indo-violet/indo-blue) of Indo-1AM loaded WT and Pak1 (T) −/− CD4 + T cell blasts, stimulated with CCL21 (200 ng/mL) for the indicated time. Results from one of three independent experiments are shown. (C) Calmodulin (CaM) was immunoprecipitated and eluted material was blotted for co-precipitation L-selectin and CaM in lysates prepared from WT and Pak1 (T) −/− T cell blasts. Whole-cell lysates were blotted for CaM. Results from two independent experiments are shown. (D) Analysis of soluble L-selectin in the supernatant of WT and Pak1 (T) −/− CD4 + T cell blasts pre-treated for 20 min with DMSO (1:1,000), BAPTA-AM 10 μM and EGTA 2 mM, or TMI1 (0.5 μM), and incubated for 30 min in the presence of 100 ng/mL CCL21. (E) Left, L-selectin surface marker expression as measured by flow cytometry from WT and Pak1 (T) −/− CD4 + T cell blasts cultured with or without TMI1(10 μM) for 6 days. The results are representative of four experiments. Right, bar chart shows MFI (%) of L-selectin in WT and Pak1 (T) −/− blast T cells. (F) Percentage of WT, Pak1 (T) −/− and Pak1 (T) −/− treated with TMI1 cells per field of view. Statistical analysis: unpaired Student's t -test (A) ; ANOVA between selected columns with Sidak's multiple comparison test (D) ; ANOVA with Tukey's multiple comparison test (E,F) . ns , not significant; * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Chick Chorioallantoic Membrane Assay, Immunoprecipitation, Incubation, Marker, Expressing, Flow Cytometry, Cytometry, Cell Culture

    4) Product Images from "TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis"

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127480

    Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].
    Figure Legend Snippet: Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].

    Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Microscopy, Transfection

    Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .
    Figure Legend Snippet: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.
    Figure Legend Snippet: TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.

    Techniques Used: Over Expression, Expressing, Marker, Fluorescence, Western Blot, Flow Cytometry, Cytometry

    Related Articles

    Incubation:

    Article Title: Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7
    Article Snippet: .. Where indicated, CD4+ T blast cells were incubated for 20 min with DMSO (1:1,000), 10 μM BAPTA-AM (Invitrogen) and 2 mM EGTA or 0.5 μM of the ADAM17 inhibitor TMI1 (Tocris Bioscience). ..

    other:

    Article Title: Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila
    Article Snippet: All nociceptin isoforms, thapsigargin, J-113397, and EGTA, were purchased from Tocris Biosciences, Bristol, UK.

    Article Title: On the Mechanism of Synaptic Depression Induced by CaMKIIN, an Endogenous Inhibitor of CaMKII
    Article Snippet: N-methyl-D-aspartic acid (NMDA) was obtained from Sigma Aldrich, and Anisomycin, EGTA, kynurenic acid, thapsigargin and MG 132 were from Tocris Bioscience.

    Fluorescence:

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis
    Article Snippet: Cells were washed once and resuspended in calcium and magnesium free HBSS (Invitrogen # 14175) with 1% FCS. .. To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences). .. Values were plotted as the ratio of fluorescence at Ca2+ -bound Indo-1 AM to that of Ca2+ -free Indo-1 AM.

    Flow Cytometry:

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis
    Article Snippet: Cells were washed once and resuspended in calcium and magnesium free HBSS (Invitrogen # 14175) with 1% FCS. .. To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences). .. Values were plotted as the ratio of fluorescence at Ca2+ -bound Indo-1 AM to that of Ca2+ -free Indo-1 AM.

    Cytometry:

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis
    Article Snippet: Cells were washed once and resuspended in calcium and magnesium free HBSS (Invitrogen # 14175) with 1% FCS. .. To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences). .. Values were plotted as the ratio of fluorescence at Ca2+ -bound Indo-1 AM to that of Ca2+ -free Indo-1 AM.

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  • egta  (Tocris)
    94
    Tocris egta
    Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM <t>EGTA</t> or 10 μM <t>BAPTA-AM,</t> for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P
    Egta, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2021-03
    94/100 stars
      Buy from Supplier

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    Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM EGTA or 10 μM BAPTA-AM, for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia

    doi: 10.1186/s12906-015-0741-1

    Figure Lengend Snippet: Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM EGTA or 10 μM BAPTA-AM, for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P

    Article Snippet: BAPTA-AM and EGTA were purchased from Tocris Bioscience (Ellisville, MO, USA).

    Techniques: Fluorescence

    Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations ( n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Journal: Journal of Virology

    Article Title: Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

    doi: 10.1128/JVI.00426-20

    Figure Lengend Snippet: Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations ( n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Article Snippet: Calcium chelators BAPTA-AM and EGTA were purchased from Tocris and VWR, respectively.

    Techniques: Infection, Luciferase, Activity Assay

    Pak1 regulates L-selectin shedding in activated CD4 + T cells. (A) Analysis by ELISA of soluble L-selectin in the supernatants of WT and Pak1 (T) −/− T cell blasts. Data were pooled from six independent experiments. (B) Representative time-dependent calcium flux (indo-violet/indo-blue) of Indo-1AM loaded WT and Pak1 (T) −/− CD4 + T cell blasts, stimulated with CCL21 (200 ng/mL) for the indicated time. Results from one of three independent experiments are shown. (C) Calmodulin (CaM) was immunoprecipitated and eluted material was blotted for co-precipitation L-selectin and CaM in lysates prepared from WT and Pak1 (T) −/− T cell blasts. Whole-cell lysates were blotted for CaM. Results from two independent experiments are shown. (D) Analysis of soluble L-selectin in the supernatant of WT and Pak1 (T) −/− CD4 + T cell blasts pre-treated for 20 min with DMSO (1:1,000), BAPTA-AM 10 μM and EGTA 2 mM, or TMI1 (0.5 μM), and incubated for 30 min in the presence of 100 ng/mL CCL21. (E) Left, L-selectin surface marker expression as measured by flow cytometry from WT and Pak1 (T) −/− CD4 + T cell blasts cultured with or without TMI1(10 μM) for 6 days. The results are representative of four experiments. Right, bar chart shows MFI (%) of L-selectin in WT and Pak1 (T) −/− blast T cells. (F) Percentage of WT, Pak1 (T) −/− and Pak1 (T) −/− treated with TMI1 cells per field of view. Statistical analysis: unpaired Student's t -test (A) ; ANOVA between selected columns with Sidak's multiple comparison test (D) ; ANOVA with Tukey's multiple comparison test (E,F) . ns , not significant; * P

    Journal: Frontiers in Immunology

    Article Title: Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7

    doi: 10.3389/fimmu.2019.00370

    Figure Lengend Snippet: Pak1 regulates L-selectin shedding in activated CD4 + T cells. (A) Analysis by ELISA of soluble L-selectin in the supernatants of WT and Pak1 (T) −/− T cell blasts. Data were pooled from six independent experiments. (B) Representative time-dependent calcium flux (indo-violet/indo-blue) of Indo-1AM loaded WT and Pak1 (T) −/− CD4 + T cell blasts, stimulated with CCL21 (200 ng/mL) for the indicated time. Results from one of three independent experiments are shown. (C) Calmodulin (CaM) was immunoprecipitated and eluted material was blotted for co-precipitation L-selectin and CaM in lysates prepared from WT and Pak1 (T) −/− T cell blasts. Whole-cell lysates were blotted for CaM. Results from two independent experiments are shown. (D) Analysis of soluble L-selectin in the supernatant of WT and Pak1 (T) −/− CD4 + T cell blasts pre-treated for 20 min with DMSO (1:1,000), BAPTA-AM 10 μM and EGTA 2 mM, or TMI1 (0.5 μM), and incubated for 30 min in the presence of 100 ng/mL CCL21. (E) Left, L-selectin surface marker expression as measured by flow cytometry from WT and Pak1 (T) −/− CD4 + T cell blasts cultured with or without TMI1(10 μM) for 6 days. The results are representative of four experiments. Right, bar chart shows MFI (%) of L-selectin in WT and Pak1 (T) −/− blast T cells. (F) Percentage of WT, Pak1 (T) −/− and Pak1 (T) −/− treated with TMI1 cells per field of view. Statistical analysis: unpaired Student's t -test (A) ; ANOVA between selected columns with Sidak's multiple comparison test (D) ; ANOVA with Tukey's multiple comparison test (E,F) . ns , not significant; * P

    Article Snippet: Where indicated, CD4+ T blast cells were incubated for 20 min with DMSO (1:1,000), 10 μM BAPTA-AM (Invitrogen) and 2 mM EGTA or 0.5 μM of the ADAM17 inhibitor TMI1 (Tocris Bioscience).

    Techniques: Enzyme-linked Immunosorbent Assay, Chick Chorioallantoic Membrane Assay, Immunoprecipitation, Incubation, Marker, Expressing, Flow Cytometry, Cytometry, Cell Culture

    Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].

    Journal: PLoS ONE

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    doi: 10.1371/journal.pone.0127480

    Figure Lengend Snippet: Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].

    Article Snippet: To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences).

    Techniques: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Microscopy, Transfection

    Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .

    Journal: PLoS ONE

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    doi: 10.1371/journal.pone.0127480

    Figure Lengend Snippet: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .

    Article Snippet: To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry

    TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.

    Journal: PLoS ONE

    Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    doi: 10.1371/journal.pone.0127480

    Figure Lengend Snippet: TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.

    Article Snippet: To measure intracellular store calcium flux cells were treated with 1mM EGTA and subsequently with thapsigargin, ionomycin, or m-3m3FBS (Tocris Bioscience) and changes in fluorescence intensity were monitored on an LSRII flow cytometer (BD Biosciences).

    Techniques: Over Expression, Expressing, Marker, Fluorescence, Western Blot, Flow Cytometry, Cytometry