bapta am  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    BAPTA AM
    Description:
    BAPTA AM inhibits free radical mediated toxicity enhances apoptosis in non neuronal cells and protects neurons from ischemic damage It regulates ion channels and blocks neuronal Ca2 activated K channel currents BAPTA AM maintains intracellular Ca2 homeostasis
    Catalog Number:
    a1076
    Price:
    None
    Applications:
    BAPTA-AM has been used :. to reduce the elevation of cleaved caspase-3. to block cell death, inhibit ROS (reactive oxygen species) production and inhibit caspase-8 activation. to exclude the effect of changes in intracellular Ca2+ ([Ca2+]i) concentration during seizure induction. to eliminate the increase of [Ca2+]i induced by 6-hydroxydopamine (6-OHDA)
    Buy from Supplier


    Structured Review

    Millipore bapta am
    BAPTA AM
    BAPTA AM inhibits free radical mediated toxicity enhances apoptosis in non neuronal cells and protects neurons from ischemic damage It regulates ion channels and blocks neuronal Ca2 activated K channel currents BAPTA AM maintains intracellular Ca2 homeostasis
    https://www.bioz.com/result/bapta am/product/Millipore
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    bapta am - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The FcεRI signaling cascade and integrin trafficking converge at patterned ligand surfaces"

    Article Title: The FcεRI signaling cascade and integrin trafficking converge at patterned ligand surfaces

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-03-0208

    β1-Integrin coclusters with IgE-FcεRI on patterned DNP-SLB in an active process that depends on actin polymerization and intracellular Ca 2+ . (A) Sensitized RBL cells were labeled with anti-β1 mAb (green) after incubation at 37°C on patterned features of DNP-SLB (red, left pair) or DNP-BSA (red, right pair). (B) Same as left pair in A, except that cells were pretreated with 2 µM CytoD. (C) Same as left pair in A, except that cells were incubated on patterned DNP-SLB surfaces in the presence of 1.5 µM RGD peptide. (D) Sensitized cells incubated at 4°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (E) Sensitized cells pretreated with 5 µM BAPTA-AM, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (F) Sensitized cells pretreated with 10 µg/ml anti-α4 mAb, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (G) Radial analysis for samples represented in A–C (carried out as described in the legend to Figure 1 ) quantify β1-integrin recruitment to DNP-SLB features. N = 30 cells from three independent experiments for each sample type. Error bars represent SEM; statistical significance: **, p ≤ 0.01. Scale bars: 10 µm.
    Figure Legend Snippet: β1-Integrin coclusters with IgE-FcεRI on patterned DNP-SLB in an active process that depends on actin polymerization and intracellular Ca 2+ . (A) Sensitized RBL cells were labeled with anti-β1 mAb (green) after incubation at 37°C on patterned features of DNP-SLB (red, left pair) or DNP-BSA (red, right pair). (B) Same as left pair in A, except that cells were pretreated with 2 µM CytoD. (C) Same as left pair in A, except that cells were incubated on patterned DNP-SLB surfaces in the presence of 1.5 µM RGD peptide. (D) Sensitized cells incubated at 4°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (E) Sensitized cells pretreated with 5 µM BAPTA-AM, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (F) Sensitized cells pretreated with 10 µg/ml anti-α4 mAb, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (G) Radial analysis for samples represented in A–C (carried out as described in the legend to Figure 1 ) quantify β1-integrin recruitment to DNP-SLB features. N = 30 cells from three independent experiments for each sample type. Error bars represent SEM; statistical significance: **, p ≤ 0.01. Scale bars: 10 µm.

    Techniques Used: Labeling, Incubation

    2) Product Images from "Novel regulation and dynamics of myosin-II activation during epidermal wound responses"

    Article Title: Novel regulation and dynamics of myosin-II activation during epidermal wound responses

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2010.01.024

    Signaling pathways important for acute myosin II activation in response to scratch-wounding Keratinocytes were allowed to form confluent monolayers, then subjected to scratch wounding. Parallel samples were harvested at each time point either with no inhibitor (black lines), or with ( A ) the MLCK inhibitor ML-7, or with ( B ) the ROCK inhibitor Y-27632, indicated with red lines. Sample western blots are presented; graphs represent average of three independent experiments for each series. ( C-F ) Upstream pathways involved in acute myosin II activation in response to scratch-wounding. Parallel samples were harvested at each time point either with no inhibitor (black lines), or with ( C ) the EGFR kinase inhibitor AG-1478, or with ( D ) the ERK1/2 inhibitor PD-098059, ( E ) the p38-MAPK inhibitor SB-202190, or ( F ) the cytosolic calcium chelator BAPTA-AM. Sample western blots are presented, graphs represent average of three independent experiments for each series.
    Figure Legend Snippet: Signaling pathways important for acute myosin II activation in response to scratch-wounding Keratinocytes were allowed to form confluent monolayers, then subjected to scratch wounding. Parallel samples were harvested at each time point either with no inhibitor (black lines), or with ( A ) the MLCK inhibitor ML-7, or with ( B ) the ROCK inhibitor Y-27632, indicated with red lines. Sample western blots are presented; graphs represent average of three independent experiments for each series. ( C-F ) Upstream pathways involved in acute myosin II activation in response to scratch-wounding. Parallel samples were harvested at each time point either with no inhibitor (black lines), or with ( C ) the EGFR kinase inhibitor AG-1478, or with ( D ) the ERK1/2 inhibitor PD-098059, ( E ) the p38-MAPK inhibitor SB-202190, or ( F ) the cytosolic calcium chelator BAPTA-AM. Sample western blots are presented, graphs represent average of three independent experiments for each series.

    Techniques Used: Activation Assay, Western Blot

    3) Product Images from "The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes"

    Article Title: The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004520

    Crosstalk between cAMP and Ca 2+ in P. falciparum merozoites. A) Ca 2+ does not regulate cytosolic cAMP levels. P. falciparum merozoites were transferred from IC to EC buffer with or without treatment with BAPTA-AM or U73122. Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca 2+ is inhibited by ACβ inhibitor KH7. P. falciparum merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca 2+ levels in P. falciparum merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca 2+ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca 2+ . P. falciparum PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca 2+ levels rise normally following transfer of P. falciparum merozoites from IC to EC buffer. Cytosolic Ca 2+ levels do not rise when P. falciparum PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca 2+ levels in merozoites. D) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca 2+ stimulated by Epac agonist. E) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca 2+ stimulated by Epac agonist. F) Cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca 2+ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca 2+ .
    Figure Legend Snippet: Crosstalk between cAMP and Ca 2+ in P. falciparum merozoites. A) Ca 2+ does not regulate cytosolic cAMP levels. P. falciparum merozoites were transferred from IC to EC buffer with or without treatment with BAPTA-AM or U73122. Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca 2+ is inhibited by ACβ inhibitor KH7. P. falciparum merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca 2+ levels in P. falciparum merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca 2+ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca 2+ . P. falciparum PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca 2+ levels rise normally following transfer of P. falciparum merozoites from IC to EC buffer. Cytosolic Ca 2+ levels do not rise when P. falciparum PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca 2+ levels in merozoites. D) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca 2+ stimulated by Epac agonist. E) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca 2+ stimulated by Epac agonist. F) Cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca 2+ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca 2+ .

    Techniques Used: Flow Cytometry, Cytometry, Planar Chromatography

    4) Product Images from "Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells"

    Article Title: Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-014-0190-7

    Induction of endoplasmic reticulum (ER)-stress markers by blockage of mGluR5. (A) Immunoblot analyses to detect GRP78 protein were performed on lysates of BV-2 cells that had been treated with 100 μM MPEP for the indicated time period. β-actin served as a loading control. (B) Densitometric analysis of GRP78 band normalized against β-actin. (C) Cells were preincubated for 1 hour with 1 μM BAPTA-AM or 1 mM sodium phenylbutyrate (PBA), followed by further incubation with 100 μM MPEP for 24 hours. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR. (D) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP for 2 hours and GRP78 protein levels were analyzed by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. (F) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR and mRNA expression levels were normalized to GAPDH control. Results represent mean ± SD of three independent experiments. Significance was analyzed by one-way ANOVA followed by Tukey’s tests. ** P
    Figure Legend Snippet: Induction of endoplasmic reticulum (ER)-stress markers by blockage of mGluR5. (A) Immunoblot analyses to detect GRP78 protein were performed on lysates of BV-2 cells that had been treated with 100 μM MPEP for the indicated time period. β-actin served as a loading control. (B) Densitometric analysis of GRP78 band normalized against β-actin. (C) Cells were preincubated for 1 hour with 1 μM BAPTA-AM or 1 mM sodium phenylbutyrate (PBA), followed by further incubation with 100 μM MPEP for 24 hours. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR. (D) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP for 2 hours and GRP78 protein levels were analyzed by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. (F) Cells were pre-exposed for 1 hour with 1 mM AICAR or 1 μM compound C, followed by a further incubation with 100 μM MPEP. Expression of CHOP, GRP78 and GRP94 mRNA were measured by quantitative PCR and mRNA expression levels were normalized to GAPDH control. Results represent mean ± SD of three independent experiments. Significance was analyzed by one-way ANOVA followed by Tukey’s tests. ** P

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction

    Prevention of cellular stress and inflammatory mediators by interference with intracellular signaling. BV-2 cells were pretreated for 1 hour with 1 μM compound C, 1 μM BAPTA-AM or 5 μM U-73122, or for 6 hours with pertussis toxin (PTX) (100 ng/mL) prior to incubation with 100 μM MPEP for 24 hours. (A) Intracellular reactive oxygen species (ROS) levels were determined using dihydroethidium (DHE). (B) Mitochondrial mass was measured using MitoTracker®Red CMXRos. (C) Mitochondrial superoxide levels were detected using MitoSOX Red. The mRNA expression of inducible oxygen species (iNOS) (D) and IL-6 (E) were determined by quantitative PCR and values were normalized to GAPDH mRNA control. (F) IL-6 protein expression was measured by ELISA. Results represent the mean ± SD of at least three independent experiments. Significance was tested by one-way ANOVA followed by Tukey’s test. * P
    Figure Legend Snippet: Prevention of cellular stress and inflammatory mediators by interference with intracellular signaling. BV-2 cells were pretreated for 1 hour with 1 μM compound C, 1 μM BAPTA-AM or 5 μM U-73122, or for 6 hours with pertussis toxin (PTX) (100 ng/mL) prior to incubation with 100 μM MPEP for 24 hours. (A) Intracellular reactive oxygen species (ROS) levels were determined using dihydroethidium (DHE). (B) Mitochondrial mass was measured using MitoTracker®Red CMXRos. (C) Mitochondrial superoxide levels were detected using MitoSOX Red. The mRNA expression of inducible oxygen species (iNOS) (D) and IL-6 (E) were determined by quantitative PCR and values were normalized to GAPDH mRNA control. (F) IL-6 protein expression was measured by ELISA. Results represent the mean ± SD of at least three independent experiments. Significance was tested by one-way ANOVA followed by Tukey’s test. * P

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Activation of phospholipase C (PLC) upon inhibition of mGluR5. (A) Intracellular Ca 2+ concentrations were determined by measuring the fluorescence intensity of the fluorochrome Fluo-4 AM. BV-2 cells were pretreated for 1 hour with 5 μM U-73122 prior to the addition of 100 μM MPEP. Relative changes in the Ca 2+ concentrations detected by Fluo-4 AM were analyzed after 15 minutes. Results are expressed as ΔF/F (in %), with F being the baseline fluorescence and ΔF the variation of fluorescence. Data represent the mean ± SD. (B) Cells were incubated for 24 hours with 100 μM MPEP in the presence or absence of 5 μM of the phospholipase C inhibitor U-73122, followed by determination of mRNA expression of CHOP, GRP78 and GRP95 by quantitative PCR. Results were normalized to GAPDH mRNA control. Results represent the mean ± SD from three independent experiments, each performed in triplicate. (C) , BV-2 cells were incubated with 100 μM MPEP for 2 hours with or without pretreatment for 1 hour with 5 μM U-73122, followed by determination of PLC activity. PLC activity (mU/μg total protein) is given as the mean ± SD from three independent experiments. (D) BV-2 cells were incubated with 100 μM MPEP in the absence or presence of 5 μM PLC inhibitor U-73122 or 1 μM Ca 2+ chelator BAPTA-AM, followed by the determination of GRP78 protein expression by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. ** P
    Figure Legend Snippet: Activation of phospholipase C (PLC) upon inhibition of mGluR5. (A) Intracellular Ca 2+ concentrations were determined by measuring the fluorescence intensity of the fluorochrome Fluo-4 AM. BV-2 cells were pretreated for 1 hour with 5 μM U-73122 prior to the addition of 100 μM MPEP. Relative changes in the Ca 2+ concentrations detected by Fluo-4 AM were analyzed after 15 minutes. Results are expressed as ΔF/F (in %), with F being the baseline fluorescence and ΔF the variation of fluorescence. Data represent the mean ± SD. (B) Cells were incubated for 24 hours with 100 μM MPEP in the presence or absence of 5 μM of the phospholipase C inhibitor U-73122, followed by determination of mRNA expression of CHOP, GRP78 and GRP95 by quantitative PCR. Results were normalized to GAPDH mRNA control. Results represent the mean ± SD from three independent experiments, each performed in triplicate. (C) , BV-2 cells were incubated with 100 μM MPEP for 2 hours with or without pretreatment for 1 hour with 5 μM U-73122, followed by determination of PLC activity. PLC activity (mU/μg total protein) is given as the mean ± SD from three independent experiments. (D) BV-2 cells were incubated with 100 μM MPEP in the absence or presence of 5 μM PLC inhibitor U-73122 or 1 μM Ca 2+ chelator BAPTA-AM, followed by the determination of GRP78 protein expression by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. ** P

    Techniques Used: Activation Assay, Planar Chromatography, Inhibition, Fluorescence, Incubation, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

    5) Product Images from "PKD2 and RSK1 Regulate Integrin β4 Phosphorylation at Threonine 1736"

    Article Title: PKD2 and RSK1 Regulate Integrin β4 Phosphorylation at Threonine 1736

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143357

    PMA- and EGF-stimulated phosphorylation of β4-T1736 is dependent on PKC and MAPK, respectively. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and then treated for 30 min with different concentrations of Gӧ6983 (100 nM-1 μM) or UO126 (2–10 μM). Subsequently the cells were stimulated with EGF or PMA for 10 min. Cell lysates were analyzed by immunoblotting with antibodies as indicated. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of β4.
    Figure Legend Snippet: PMA- and EGF-stimulated phosphorylation of β4-T1736 is dependent on PKC and MAPK, respectively. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and then treated for 30 min with different concentrations of Gӧ6983 (100 nM-1 μM) or UO126 (2–10 μM). Subsequently the cells were stimulated with EGF or PMA for 10 min. Cell lysates were analyzed by immunoblotting with antibodies as indicated. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of β4.

    Techniques Used:

    PI3K and mTOR are not required for EGF-mediated β4 phosphorylation. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM and/or different concentrations of the mTOR kinase inhibitor AZD 8055 (0,8–100 nM) or the PI3K inhibitor GDC-0941 (8 nM- 1 μM) and subsequently stimulated with EGF or left unstimulated. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 after normalization to the level of α-tubulin.
    Figure Legend Snippet: PI3K and mTOR are not required for EGF-mediated β4 phosphorylation. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM and/or different concentrations of the mTOR kinase inhibitor AZD 8055 (0,8–100 nM) or the PI3K inhibitor GDC-0941 (8 nM- 1 μM) and subsequently stimulated with EGF or left unstimulated. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 after normalization to the level of α-tubulin.

    Techniques Used:

    EGF- but not PMA-stimulated phosphorylation of β4 at T1736 and S1364 depends on RSK1/2. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and/or BI-D1870 for 30 min at the indicated concentrations or left untreated and then stimulated with EGF or PMA. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of α-tubulin.
    Figure Legend Snippet: EGF- but not PMA-stimulated phosphorylation of β4 at T1736 and S1364 depends on RSK1/2. PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and/or BI-D1870 for 30 min at the indicated concentrations or left untreated and then stimulated with EGF or PMA. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of α-tubulin.

    Techniques Used:

    6) Product Images from "Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation"

    Article Title: Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation

    Journal: Apoptosis

    doi: 10.1007/s10495-015-1095-3

    Mcl-1 enforced expression rescue ovarian carcinoma cells from apoptosis triggered by BAPTA-AM/ABT-737 or W7/ABT-737 combinations. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing Mcl-1 ORF (Mcl-1) for 40 h as described in “ Materials and methods ” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. a DNA contents were studied for each condition. b Cell viability was assessed by trypan blue exclusion. c Mcl-1 expression and Caspase 3 cleavage were studied by western-blot. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing EIF4E ORF (eIF4E) for 40 h as described in “ Materials and methods ” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. d DNA contents were studied for each condition. e Cell viability was assessed by trypan blue exclusion. f Mcl-1, eIF4E expression and PARP and Caspase 3 cleavage were studied by western-blot
    Figure Legend Snippet: Mcl-1 enforced expression rescue ovarian carcinoma cells from apoptosis triggered by BAPTA-AM/ABT-737 or W7/ABT-737 combinations. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing Mcl-1 ORF (Mcl-1) for 40 h as described in “ Materials and methods ” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. a DNA contents were studied for each condition. b Cell viability was assessed by trypan blue exclusion. c Mcl-1 expression and Caspase 3 cleavage were studied by western-blot. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing EIF4E ORF (eIF4E) for 40 h as described in “ Materials and methods ” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. d DNA contents were studied for each condition. e Cell viability was assessed by trypan blue exclusion. f Mcl-1, eIF4E expression and PARP and Caspase 3 cleavage were studied by western-blot

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot

    Calcium chelation combined with ABT-737 leads to apoptosis in ovarian carcinoma. a Real time analysis of cellular cytotoxicity of ABT-737/BAPTA-AM combination. Histogram was obtained using the xCELLigence System as described in “ Materials and methods ” section. Cells were grown for 24 h and then treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM. Cell index was recorded every 2 h, with displayed standard error bars. IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM for 6 and 24 h. b Morphological features and c DNA contents were studied for each condition. d Cell viability was assessed by trypan blue exclusion at 24 h. e PARP and caspase 3 cleavages were studied by western-blot. Data are representative of three independent experiments
    Figure Legend Snippet: Calcium chelation combined with ABT-737 leads to apoptosis in ovarian carcinoma. a Real time analysis of cellular cytotoxicity of ABT-737/BAPTA-AM combination. Histogram was obtained using the xCELLigence System as described in “ Materials and methods ” section. Cells were grown for 24 h and then treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM. Cell index was recorded every 2 h, with displayed standard error bars. IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM for 6 and 24 h. b Morphological features and c DNA contents were studied for each condition. d Cell viability was assessed by trypan blue exclusion at 24 h. e PARP and caspase 3 cleavages were studied by western-blot. Data are representative of three independent experiments

    Techniques Used: Western Blot

    7) Product Images from "The role of cGMP in the regulation of rabbit airway ciliary beat frequency"

    Article Title: The role of cGMP in the regulation of rabbit airway ciliary beat frequency

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2003.041707

    The combined effect of buffering [Ca 2+ ] i with BAPTA and inhibiting PKG activity with KT5823 on the response to Br-cGMP A and B , two representative cells illustrate the combined effect of the pretreatment of cells with KT5823 and BAPTA AM on the changes in CBF (black dots) and [Ca 2+ ] i (grey lines) induced by 100 μM Br-cGMP (bar). KT5823 and BAPTA AM treatment prevented any significant increases in the [Ca 2+ ] i and CBF in response to Br-cGMP. The basal CBF of the cells was 17.7 Hz ( A ) and 12.5 Hz ( B ). C and D , summaries of the combined effects of KT5823 and BAPTA AM on the initial ( C ) and sustained ( D ) increases in CBF induced by 100 μM Br-cGMP. The increase in the initial CBF (1.09 ± 0.03, n = 7) induced by Br-cGMP in the presence of KT5823 and BAPTA AM was significantly lower than the increase induced in cells without treatment (1.32 ± 0.08, n = 6, * P
    Figure Legend Snippet: The combined effect of buffering [Ca 2+ ] i with BAPTA and inhibiting PKG activity with KT5823 on the response to Br-cGMP A and B , two representative cells illustrate the combined effect of the pretreatment of cells with KT5823 and BAPTA AM on the changes in CBF (black dots) and [Ca 2+ ] i (grey lines) induced by 100 μM Br-cGMP (bar). KT5823 and BAPTA AM treatment prevented any significant increases in the [Ca 2+ ] i and CBF in response to Br-cGMP. The basal CBF of the cells was 17.7 Hz ( A ) and 12.5 Hz ( B ). C and D , summaries of the combined effects of KT5823 and BAPTA AM on the initial ( C ) and sustained ( D ) increases in CBF induced by 100 μM Br-cGMP. The increase in the initial CBF (1.09 ± 0.03, n = 7) induced by Br-cGMP in the presence of KT5823 and BAPTA AM was significantly lower than the increase induced in cells without treatment (1.32 ± 0.08, n = 6, * P

    Techniques Used: Activity Assay

    The effect of buffering the [Ca 2+ ] i with BAPTA in response to 100 μM Br-cGMP A , a representative response of a ciliated cell pretreated with 20 μM BAPTA AM and exposed to Br-cGMP (bar). The BAPTA buffering prevented any changes in [Ca 2+ ] i (grey line). However, the CBF (black dots) still increased (to an initial normalized CBF = 1.36) and remained sustained (at a normalized CBF = 1.32). Transient increases in [Ca 2+ ] i or CBF did not occur. The basal CBF was 12.1 Hz. B and C , the effect of buffering the [Ca 2+ ] i with BAPTA on the initial ( B ) and sustained ( C ) changes in CBF induced by Br-cGMP. In control experiments, without BAPTA treatment, the initial and sustained increases in CBF induced by Br-cGMP were 1.28 ± 0.05 ( n = 11) and 1.24 ± 0.04 ( n = 11) respectively. These changes were indistinguishable ( P > 0.05) from the initial (1.32 ± 0.08, n = 6) and sustained (1.23 ± 0.05, n = 6) increases in CBF induced by Br-cGMP in the presence of BAPTA. The basal CBFs for cells with (12.4 ± 1.0, n = 6) or without BAPTA treatment (12.4 ± 0.6, n = 11) were similar ( P > 0.05).
    Figure Legend Snippet: The effect of buffering the [Ca 2+ ] i with BAPTA in response to 100 μM Br-cGMP A , a representative response of a ciliated cell pretreated with 20 μM BAPTA AM and exposed to Br-cGMP (bar). The BAPTA buffering prevented any changes in [Ca 2+ ] i (grey line). However, the CBF (black dots) still increased (to an initial normalized CBF = 1.36) and remained sustained (at a normalized CBF = 1.32). Transient increases in [Ca 2+ ] i or CBF did not occur. The basal CBF was 12.1 Hz. B and C , the effect of buffering the [Ca 2+ ] i with BAPTA on the initial ( B ) and sustained ( C ) changes in CBF induced by Br-cGMP. In control experiments, without BAPTA treatment, the initial and sustained increases in CBF induced by Br-cGMP were 1.28 ± 0.05 ( n = 11) and 1.24 ± 0.04 ( n = 11) respectively. These changes were indistinguishable ( P > 0.05) from the initial (1.32 ± 0.08, n = 6) and sustained (1.23 ± 0.05, n = 6) increases in CBF induced by Br-cGMP in the presence of BAPTA. The basal CBFs for cells with (12.4 ± 1.0, n = 6) or without BAPTA treatment (12.4 ± 0.6, n = 11) were similar ( P > 0.05).

    Techniques Used:

    8) Product Images from "Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells"

    Article Title: Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

    Journal: American Journal of Physiology. Renal Physiology

    doi: 10.1152/ajprenal.00108.2008

    Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P
    Figure Legend Snippet: Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P

    Techniques Used: Fluorescence, Produced

    Distribution of BGT1 protein (green) in MDCK cell monolayers is primarily in the plasma membrane after hypertonic stress (500 mosmol/kgH 2 O) for 24 h ( B ) in marked contrast to cells maintained in isotonic medium ( A ). Treatment of hypertonic cells for 30 min with 0.1 mM ATP ( C ) or 0.1 mM adenosine ( D ) produced punctuate green fluorescence (arrows) in the cytoplasm of many cells, consistent with endocytic internalization of BGT1 protein. In contrast, almost no intracellular punctuate green fluorescence was observed when BAPTA-loaded cells were treated with 0.1 mM ATP ( F ), compared with the action of ATP on control cells ( E ). Cells in A–D were fixed in paraformaldehyde, permeabilized, and BGT1 was detected with BGT1 antibody from Proteintech. Cells in E–F were methanol fixed and BGT1 was detected with antibody provided by Dr. H. M. Kwon. Nucleic acids and nuclei were counterstained with propidium iodide (red). Bar = 20 μm.
    Figure Legend Snippet: Distribution of BGT1 protein (green) in MDCK cell monolayers is primarily in the plasma membrane after hypertonic stress (500 mosmol/kgH 2 O) for 24 h ( B ) in marked contrast to cells maintained in isotonic medium ( A ). Treatment of hypertonic cells for 30 min with 0.1 mM ATP ( C ) or 0.1 mM adenosine ( D ) produced punctuate green fluorescence (arrows) in the cytoplasm of many cells, consistent with endocytic internalization of BGT1 protein. In contrast, almost no intracellular punctuate green fluorescence was observed when BAPTA-loaded cells were treated with 0.1 mM ATP ( F ), compared with the action of ATP on control cells ( E ). Cells in A–D were fixed in paraformaldehyde, permeabilized, and BGT1 was detected with BGT1 antibody from Proteintech. Cells in E–F were methanol fixed and BGT1 was detected with antibody provided by Dr. H. M. Kwon. Nucleic acids and nuclei were counterstained with propidium iodide (red). Bar = 20 μm.

    Techniques Used: Produced, Fluorescence

    9) Product Images from "A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation"

    Article Title: A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006044

    Importance of intracellular signaling cascades in the pMV-absorption. (A) Wild type (WT), CD28 −/− , and LFA-1 −/− 2C T cells, pre- treated with the respective mAbs, were cultured with or without the peptide-loaded L d B7-1ICAM-1 pMVs, as indicated, followed by mAb staining for B7-1 and flow cytometric analysis. Mean fluorescence intensities (MFIs) of B7-1 staining relative to that of WT 2C T cells, pre-treated with control mAb and cultured with the QL9-loaded pMVs, were plotted. Note that P1A peptide forms complex with L d but L d /P1A complex is not recognized by 2C TCR [8] . (B) WT 2C T cells, pre-treated with the respective mAbs, were cultured with the peptide-loaded pMVs for different periods of time, as indicated, and stained for B7-1. (C) WT 2C T cells were treated with PP2 (PP), Wortmannin (WM), Pervanadate (PV), Cytochalasin D (CT), BAPTA/AM (BA), Forskolin (FS), Cyclosporin A (CC) and Nocodazole (NZ), respectively, as indicated, before culture with the QL9-loaded pMVs. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted.
    Figure Legend Snippet: Importance of intracellular signaling cascades in the pMV-absorption. (A) Wild type (WT), CD28 −/− , and LFA-1 −/− 2C T cells, pre- treated with the respective mAbs, were cultured with or without the peptide-loaded L d B7-1ICAM-1 pMVs, as indicated, followed by mAb staining for B7-1 and flow cytometric analysis. Mean fluorescence intensities (MFIs) of B7-1 staining relative to that of WT 2C T cells, pre-treated with control mAb and cultured with the QL9-loaded pMVs, were plotted. Note that P1A peptide forms complex with L d but L d /P1A complex is not recognized by 2C TCR [8] . (B) WT 2C T cells, pre-treated with the respective mAbs, were cultured with the peptide-loaded pMVs for different periods of time, as indicated, and stained for B7-1. (C) WT 2C T cells were treated with PP2 (PP), Wortmannin (WM), Pervanadate (PV), Cytochalasin D (CT), BAPTA/AM (BA), Forskolin (FS), Cyclosporin A (CC) and Nocodazole (NZ), respectively, as indicated, before culture with the QL9-loaded pMVs. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted.

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Fluorescence

    10) Product Images from "Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells"

    Article Title: Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells

    Journal: Journal of Ginseng Research

    doi: 10.1016/j.jgr.2017.01.015

    Compound K induced apoptosis via Ca 2+ mediated caspase-12 activation. (A) Cytosolic Ca 2+ levels were measured by Fura-2/AM fluorescence dye. Compound K treated 30 min after the beginning of the reading as indicated by the arrow. (B, C, D) Effect of the RyR channel antagonist (dantrolene), IP 3 R channel antagonist (2-APB), or Ca 2+ chelator (BAPTA-AM) on apoptosis was determined by testing the costaining with PI and FITC-conjugated annexin V, and the translocation of phosphatidylserine was detected by flow cytometry. Data are presented as means ± SD of three independent experiments. (E, F) Cells were pretreated with 5μM dantrolene or 2.5μM BAPTA-AM for 1 h and then treated with 15μM of compound K for 48 h. m-Calpain, caspase-12, and procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. (G) Cells were pretreated with 30μM 2-APB for 1 h and were then treated with 15μM of compound K for 48 h. Procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. *** p
    Figure Legend Snippet: Compound K induced apoptosis via Ca 2+ mediated caspase-12 activation. (A) Cytosolic Ca 2+ levels were measured by Fura-2/AM fluorescence dye. Compound K treated 30 min after the beginning of the reading as indicated by the arrow. (B, C, D) Effect of the RyR channel antagonist (dantrolene), IP 3 R channel antagonist (2-APB), or Ca 2+ chelator (BAPTA-AM) on apoptosis was determined by testing the costaining with PI and FITC-conjugated annexin V, and the translocation of phosphatidylserine was detected by flow cytometry. Data are presented as means ± SD of three independent experiments. (E, F) Cells were pretreated with 5μM dantrolene or 2.5μM BAPTA-AM for 1 h and then treated with 15μM of compound K for 48 h. m-Calpain, caspase-12, and procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. (G) Cells were pretreated with 30μM 2-APB for 1 h and were then treated with 15μM of compound K for 48 h. Procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. *** p

    Techniques Used: Activation Assay, Fluorescence, Translocation Assay, Flow Cytometry, Cytometry, Western Blot

    11) Product Images from "TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response"

    Article Title: TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20150353

    Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Activation Assay, Transmission Electron Microscopy, Concentration Assay, Fluorescence, Positive Control, Isolation

    12) Product Images from "Energy of the Lowest Unoccupied Molecular Orbital, Thiol Reactivity, and Toxicity of Three Monobrominated Water Disinfection Byproducts"

    Article Title: Energy of the Lowest Unoccupied Molecular Orbital, Thiol Reactivity, and Toxicity of Three Monobrominated Water Disinfection Byproducts

    Journal: Environmental science & technology

    doi: 10.1021/acs.est.5b05581

    Effect of the Ca 2+ -specific chelator BAPTA-AM on genomic DNA damage induced by BAA (75 μ M), BAN (60 μ M), BAM (60 μ M), EMS (3.8 mM), or H 2 O 2 (0.003%). Each genotoxin served as its 100% DNA damage concurrent positive control. Chelating
    Figure Legend Snippet: Effect of the Ca 2+ -specific chelator BAPTA-AM on genomic DNA damage induced by BAA (75 μ M), BAN (60 μ M), BAM (60 μ M), EMS (3.8 mM), or H 2 O 2 (0.003%). Each genotoxin served as its 100% DNA damage concurrent positive control. Chelating

    Techniques Used: Positive Control

    13) Product Images from "Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells"

    Article Title: Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4278

    Inhibition of calcium signaling decreases cisplatin-induced mitochondria-mediated apoptosis in HeLa cells. (A) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h and then stained with Annexin-V. Data are presented as the mean ± SD (n=3). (B) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h, and stained with Hoechst 33342. Cell morphology was observed by confocal microscopy (scale bar, 30 µm). (C) The expression of calpain, caspase-3 and cleaved caspase-3 in HeLa cells treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 h by western blotting. (D) Quantitation of calpain and cleaved caspase-3 protein levels. Data are presented as the mean ± SD (n=3). *P
    Figure Legend Snippet: Inhibition of calcium signaling decreases cisplatin-induced mitochondria-mediated apoptosis in HeLa cells. (A) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h and then stained with Annexin-V. Data are presented as the mean ± SD (n=3). (B) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h, and stained with Hoechst 33342. Cell morphology was observed by confocal microscopy (scale bar, 30 µm). (C) The expression of calpain, caspase-3 and cleaved caspase-3 in HeLa cells treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 h by western blotting. (D) Quantitation of calpain and cleaved caspase-3 protein levels. Data are presented as the mean ± SD (n=3). *P

    Techniques Used: Inhibition, Staining, Confocal Microscopy, Expressing, Western Blot, Quantitation Assay

    Inhibition of calcium signaling decreases cisplatin-induced ER stress-mediated apoptosis in HeLa cells. (A) Western blotting detection of ER stress proteins in HeLa cells treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 h. (B) Quantitation of ER stress protein levels. Data are presented as the mean ± standard deviation (n=3). *P
    Figure Legend Snippet: Inhibition of calcium signaling decreases cisplatin-induced ER stress-mediated apoptosis in HeLa cells. (A) Western blotting detection of ER stress proteins in HeLa cells treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 h. (B) Quantitation of ER stress protein levels. Data are presented as the mean ± standard deviation (n=3). *P

    Techniques Used: Inhibition, Western Blot, Quantitation Assay, Standard Deviation

    Inhibition of calcium signaling decreases the level of free Ca 2+ in the cytosol and mitochondria, and inhibits cell growth. (A) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 and 24 h. The cells were incubated with the fluorescent calcium indicator, Fluo-4/AM. Calcium concentrations in the cytosol were observed by confocal microscopy (scale bar, 40 µm). (B) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 and 24 h, and incubated with the fluorescent calcium indicator, Rhod-2. Calcium concentrations in the mitochondria were observed by confocal microscopy (scale bar, 30 µm). (C) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h. Cell viability was determined using the 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are presented as the mean ± standard deviation (n=3). *P
    Figure Legend Snippet: Inhibition of calcium signaling decreases the level of free Ca 2+ in the cytosol and mitochondria, and inhibits cell growth. (A) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 and 24 h. The cells were incubated with the fluorescent calcium indicator, Fluo-4/AM. Calcium concentrations in the cytosol were observed by confocal microscopy (scale bar, 40 µm). (B) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 12 and 24 h, and incubated with the fluorescent calcium indicator, Rhod-2. Calcium concentrations in the mitochondria were observed by confocal microscopy (scale bar, 30 µm). (C) HeLa cells were treated with cisplatin (5 µg/ml) with or without BAPTA/AM (2.5 µM) and 2-APB (100 µM) for 24 h. Cell viability was determined using the 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are presented as the mean ± standard deviation (n=3). *P

    Techniques Used: Inhibition, Incubation, Confocal Microscopy, Standard Deviation

    14) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

    Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.1166-09.2009

    ( a ) Induction of CREB-dependent gene expression by cAMP requires Ca 2+ . Stimulation of 4x CRE luciferase activity by forskolin (10 μM) or 8-Br-cAMP (3 mM) was abolished by adding 3 mM Bapta-free acid (Bapta-FA) or by pre-treating with Bapta-AM (10
    Figure Legend Snippet: ( a ) Induction of CREB-dependent gene expression by cAMP requires Ca 2+ . Stimulation of 4x CRE luciferase activity by forskolin (10 μM) or 8-Br-cAMP (3 mM) was abolished by adding 3 mM Bapta-free acid (Bapta-FA) or by pre-treating with Bapta-AM (10

    Techniques Used: Expressing, Luciferase, Activity Assay

    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
    Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Techniques Used: Plasmid Preparation

    15) Product Images from "Anti-proliferative Effects of Nucleotides on Gastric Cancer via a Novel P2Y6/SOCE/Ca2+/β-catenin Pathway"

    Article Title: Anti-proliferative Effects of Nucleotides on Gastric Cancer via a Novel P2Y6/SOCE/Ca2+/β-catenin Pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02562-x

    UTP-repressed gastric cancer cell proliferation through P2Y6-mediated Ca 2+ and β-catenin signaling. ( a – h ) UTP, UDP, spiperone and CPA does-dependently inhibited proliferation of MKN-45 and SGC-7901 GC cells. ( i , j ) Both P2Y6 receptor selective inhibitor MRS2578 (3 µM) and knockdown of P2Y6 receptors reversed UTP (20 µM)-repressed MKN-45 cell proliferation. ( k , l ) Both intracellular calcium chelater BAPTA-AM (1 µM) and SOC inhibitor 2-APB (50 µM) rescued UTP-repressed MKN-45 cell proliferation. ( m ) Over-expression β-catenin rescued UTP-repressed MKN-45 cell proliferation. *p
    Figure Legend Snippet: UTP-repressed gastric cancer cell proliferation through P2Y6-mediated Ca 2+ and β-catenin signaling. ( a – h ) UTP, UDP, spiperone and CPA does-dependently inhibited proliferation of MKN-45 and SGC-7901 GC cells. ( i , j ) Both P2Y6 receptor selective inhibitor MRS2578 (3 µM) and knockdown of P2Y6 receptors reversed UTP (20 µM)-repressed MKN-45 cell proliferation. ( k , l ) Both intracellular calcium chelater BAPTA-AM (1 µM) and SOC inhibitor 2-APB (50 µM) rescued UTP-repressed MKN-45 cell proliferation. ( m ) Over-expression β-catenin rescued UTP-repressed MKN-45 cell proliferation. *p

    Techniques Used: Over Expression

    Suppression of β-catenin by P2Y6-induced Ca 2+ signaling in GC cells. ( a – f ) UTP (20 µM), UDP (50 µM) and spiperone (10 µM) decreased β-catenin(Ser675) at 24 hours of MKN-45 and SGC-7901 cells. ( g , h ) MRS2578 (3 µM) reversed both UTP and UDP-repressed β-catenin(Ser675). ( i ) Knockdown of P2Y6 receptors reversed UTP induced decrease of β-catenin(Ser675). ( j , k ) 2-APB (50 µM) reversed UTP and spiperone-induced decrease of β-catenin(Ser675). ( l ) BAPTA (1 µM) reversed UTP induced decrease of β-catenin(Ser675).
    Figure Legend Snippet: Suppression of β-catenin by P2Y6-induced Ca 2+ signaling in GC cells. ( a – f ) UTP (20 µM), UDP (50 µM) and spiperone (10 µM) decreased β-catenin(Ser675) at 24 hours of MKN-45 and SGC-7901 cells. ( g , h ) MRS2578 (3 µM) reversed both UTP and UDP-repressed β-catenin(Ser675). ( i ) Knockdown of P2Y6 receptors reversed UTP induced decrease of β-catenin(Ser675). ( j , k ) 2-APB (50 µM) reversed UTP and spiperone-induced decrease of β-catenin(Ser675). ( l ) BAPTA (1 µM) reversed UTP induced decrease of β-catenin(Ser675).

    Techniques Used:

    16) Product Images from "Sphingosine-1-Phosphate Receptor 3 Mediates Sphingosine-1-Phosphate Induced Release of Weibel-Palade Bodies from Endothelial Cells"

    Article Title: Sphingosine-1-Phosphate Receptor 3 Mediates Sphingosine-1-Phosphate Induced Release of Weibel-Palade Bodies from Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091346

    Pharmacological inhibition of S1P-induced VWF secretion. CTRL and Y27632 treated HUVECs were incubated for indicated times with 1 μM S1P or SF medium alone (-) (A). (B) siCTRL and siS1PR1 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of 10 μM Rho kinase inhibitor Y27632 or 100 μM calcium chelator BAPTA-AM. (C) siCTRL and siS1PR3 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of Rho kinase inhibitor Y27632 or calcium inhibitor BAPTA-AM. The amount of VWF secreted in the medium was measured by ELISA; VWF secreted by unstimulated siCTRL treated cells after 45 minutes was set to 100%. Three independent experiments were performed. Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P
    Figure Legend Snippet: Pharmacological inhibition of S1P-induced VWF secretion. CTRL and Y27632 treated HUVECs were incubated for indicated times with 1 μM S1P or SF medium alone (-) (A). (B) siCTRL and siS1PR1 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of 10 μM Rho kinase inhibitor Y27632 or 100 μM calcium chelator BAPTA-AM. (C) siCTRL and siS1PR3 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of Rho kinase inhibitor Y27632 or calcium inhibitor BAPTA-AM. The amount of VWF secreted in the medium was measured by ELISA; VWF secreted by unstimulated siCTRL treated cells after 45 minutes was set to 100%. Three independent experiments were performed. Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P

    Techniques Used: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Heavy Alcohol Exposure Activates Astroglial Hemichannels and Pannexons in the Hippocampus of Adolescent Rats: Effects on Neuroinflammation and Astrocyte Arborization"

    Article Title: Heavy Alcohol Exposure Activates Astroglial Hemichannels and Pannexons in the Hippocampus of Adolescent Rats: Effects on Neuroinflammation and Astrocyte Arborization

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00472

    Hemichannel and pannexon activity induced by intermittent heavy ethanol exposure depends on p38 mitogen-activated protein kinase (MAPK)/inducible nitric oxide synthase (iNOS)/cyclooxygenases (COXs) pathways. (A–C) Averaged data of Etd uptake normalized to control conditions (dashed line) by astrocytes in the stratum oriens (A) , stratum pyramidale (B) and stratum radiatum (C) from rats after 1-week following ethanol exposure. Also shown are the effects of the following blockers applied during the Etd uptake assay: SB203580 (10 μM, p38 MAPK inhibitor), LN-6 (1 μM, iNOS inhibitor), sc-560 (40 μM, COX 1 inhibitor), ns-398 (10 μM, COX2 inhibitor) and BAPTA (10 μM, Ca 2+ chelator). # p
    Figure Legend Snippet: Hemichannel and pannexon activity induced by intermittent heavy ethanol exposure depends on p38 mitogen-activated protein kinase (MAPK)/inducible nitric oxide synthase (iNOS)/cyclooxygenases (COXs) pathways. (A–C) Averaged data of Etd uptake normalized to control conditions (dashed line) by astrocytes in the stratum oriens (A) , stratum pyramidale (B) and stratum radiatum (C) from rats after 1-week following ethanol exposure. Also shown are the effects of the following blockers applied during the Etd uptake assay: SB203580 (10 μM, p38 MAPK inhibitor), LN-6 (1 μM, iNOS inhibitor), sc-560 (40 μM, COX 1 inhibitor), ns-398 (10 μM, COX2 inhibitor) and BAPTA (10 μM, Ca 2+ chelator). # p

    Techniques Used: Activity Assay

    18) Product Images from "Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease"

    Article Title: Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI27798

    The traversal of the human BBB model by T. b. gambiense requires calcium and the activity of papain-like cysteine proteases. ( A ) Bloodstream forms of T. b. gambiense parasites (10 6 ) were incubated with human BMECs on Transwell inserts for 3 hours. The number of parasites at the lower chamber was subsequently estimated by counting in a hemacytometer. The percentage of parasites ± SEM that crossed relative to the 10 6 parasites added is shown. Experiments were performed in triplicate. To show the role of [Ca 2+ ] i , the BMECs were incubated with 15 μM BAPTA-AM for 30 minutes and washed twice prior to adding parasites. Controls were performed by adding medium containing 0.5% DMSO. ( B ) Bloodstream-form parasites (10 6 ) were added to the endothelial cells and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A . DMSO (0.5%), K11777 (20 μM), and E-64d (20 μM) were added to human BMEC monolayers immediately before the addition of parasites. ( C ) Bloodstream-form parasites (10 6 ) were added to inserts containing human BMECs or empty inserts (No BMECs) and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A . DMSO (0.5%) and K11777 (1–20 μM) were added to insert monolayers immediately before the addition of parasites. * P
    Figure Legend Snippet: The traversal of the human BBB model by T. b. gambiense requires calcium and the activity of papain-like cysteine proteases. ( A ) Bloodstream forms of T. b. gambiense parasites (10 6 ) were incubated with human BMECs on Transwell inserts for 3 hours. The number of parasites at the lower chamber was subsequently estimated by counting in a hemacytometer. The percentage of parasites ± SEM that crossed relative to the 10 6 parasites added is shown. Experiments were performed in triplicate. To show the role of [Ca 2+ ] i , the BMECs were incubated with 15 μM BAPTA-AM for 30 minutes and washed twice prior to adding parasites. Controls were performed by adding medium containing 0.5% DMSO. ( B ) Bloodstream-form parasites (10 6 ) were added to the endothelial cells and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A . DMSO (0.5%), K11777 (20 μM), and E-64d (20 μM) were added to human BMEC monolayers immediately before the addition of parasites. ( C ) Bloodstream-form parasites (10 6 ) were added to inserts containing human BMECs or empty inserts (No BMECs) and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A . DMSO (0.5%) and K11777 (1–20 μM) were added to insert monolayers immediately before the addition of parasites. * P

    Techniques Used: Activity Assay, Incubation

    19) Product Images from "Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis"

    Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127279

    MSU crystals enhance Ca 2+ dependent ROS production. ( A ) dTHP-1 cells were incubated with 3 μM Fluo-3/AM at 37°C for 1 hour in the dark and were stimulated with 5 μg/ml MSU. After stimulation, fluorescence emission was continuously monitored for 30 minutes and expressed as to determine relative alteration in intensity. ( B ) dTHP-1 cells were incubated for 1 hour at the dark with 10 μM DCF, or with 3 μM Fluo-3/AM (inset of the figure), and were stimulated with 5 μg/ml MSU. Ca 2+ dependence ROS generation was assessed by adding 20 μM BAPTA-AM or 3 mM EGTA 30 minutes and 15 minutes before MSU addition, respectively. Fluorescence emission was monitored at 20 minutes after stimulation. Results are expressed as mean ± SD of arbitrary fluorescence units performed in triplicate and are representative of three separate experiments. * p
    Figure Legend Snippet: MSU crystals enhance Ca 2+ dependent ROS production. ( A ) dTHP-1 cells were incubated with 3 μM Fluo-3/AM at 37°C for 1 hour in the dark and were stimulated with 5 μg/ml MSU. After stimulation, fluorescence emission was continuously monitored for 30 minutes and expressed as to determine relative alteration in intensity. ( B ) dTHP-1 cells were incubated for 1 hour at the dark with 10 μM DCF, or with 3 μM Fluo-3/AM (inset of the figure), and were stimulated with 5 μg/ml MSU. Ca 2+ dependence ROS generation was assessed by adding 20 μM BAPTA-AM or 3 mM EGTA 30 minutes and 15 minutes before MSU addition, respectively. Fluorescence emission was monitored at 20 minutes after stimulation. Results are expressed as mean ± SD of arbitrary fluorescence units performed in triplicate and are representative of three separate experiments. * p

    Techniques Used: Incubation, Fluorescence

    20) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Induces Rapid Release of Angiopoietin-2 from Endothelial Cells"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces Rapid Release of Angiopoietin-2 from Endothelial Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.03303-12

    Ca 2+ chelators and Ca 2+ channel blockers inhibit KSHV-induced Ang-2 release. Identical numbers of HUVECs were pretreated with the Ca 2+ chelators BAPTA (100 μM), BAPTA-AM (100 μM), and EGTA (100 μM); the Ca 2+ channel blockers nifedipine
    Figure Legend Snippet: Ca 2+ chelators and Ca 2+ channel blockers inhibit KSHV-induced Ang-2 release. Identical numbers of HUVECs were pretreated with the Ca 2+ chelators BAPTA (100 μM), BAPTA-AM (100 μM), and EGTA (100 μM); the Ca 2+ channel blockers nifedipine

    Techniques Used:

    21) Product Images from "Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore"

    Article Title: Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

    Journal: Autophagy

    doi: 10.4161/auto.6.7.13005

    Bnip3 induces autophagy independent of Ca 2+ , ROS and mPTP opening. (A) Starvation-induced autophagy is inhibited in the presence of 0.2 µM BAPTA-AM, 1 µM CsA or 1 mM NAC in adult myocytes (n = 3, *p
    Figure Legend Snippet: Bnip3 induces autophagy independent of Ca 2+ , ROS and mPTP opening. (A) Starvation-induced autophagy is inhibited in the presence of 0.2 µM BAPTA-AM, 1 µM CsA or 1 mM NAC in adult myocytes (n = 3, *p

    Techniques Used:

    22) Product Images from "Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes"

    Article Title: Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073678

    BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p
    Figure Legend Snippet: BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p

    Techniques Used: Expressing, Cell Culture, Irradiation, Incubation, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes"

    Article Title: Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073678

    BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p
    Figure Legend Snippet: BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p

    Techniques Used: Expressing, Cell Culture, Irradiation, Incubation, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes"

    Article Title: Violet Light Down-Regulates the Expression of Specific Differentiation Markers through Rhodopsin in Normal Human Epidermal Keratinocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073678

    BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p
    Figure Legend Snippet: BAPTA-AM and W7 had no significant effects on the expression levels of the differentiation markers in rhodopsin-over-expressed NHEKs. NHEKs were cultured and then treated with BAPTA-AM (10 mM) or W7 (40 mM) 1 hr before irradiation. The cells were incubated for 24 hr after irradiation with 410 nm wavelength light for 60 min. Q-RT-PCR was performed to assess the expression of (A) keratin-5, (B) keratin-10, (C) transglutaminase-3, and (D) filaggrin. The data are representative of at least three independent experiments. The values represent the mean ± SEM of the mRNA expression corresponding to the various genes relative to control which are normalized to human RPL13A expression (n = 3 independent cell lines in triplicate) *p

    Techniques Used: Expressing, Cell Culture, Irradiation, Incubation, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection"

    Article Title: Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000415

    FimH induces UPIIIa phosphorylation and intracellular calcium elevation. (A) PD07i cells were stimulated with either 10 µg/ml FimCH or 10 µg/ml BSA for 30 minutes, and UPIIIa was immunoprecipitated from 100 µg cell extract with anti-UPIII antibody. Following electrophoresis and blotting, immunoprecipitated proteins were probed with an anti-phosphothreonine antibody (P-Thr). Blots were subsequently re-probed with an anti-UPIII antibody to determine total protein loading (UP3) (B) PD07i cultures were loaded with 5 µM Fura-2 and imaged for 5 minutes at 340 nm and 380 nm using real-time video fluorescence microscopy. FimCH (10 µg/ml) was added after establishing baseline Ca 2+ concentrations collection for 1 min (arrow). The trace is the mean of a representative experiment of three replicates. (C) Extracellular Ca 2+ is required for FimCH-induced urothelial Ca 2+ elevation. PD07i cells were loaded with 5 µM Fura-2 and imaged for 10 minutes in nominal Ca 2+ -free medium. Cells were then exposed to 10 µg/ml FimCH, imaged for 5 minutes followed by addition of Ca 2+ to a final concentration of 2.5 mM to the buffer solution and data were acquired for another 5 min. Baseline Ca 2+ concentration is represented by the dotted line. The trace is the mean of a representative experiment of three replicates. (D) FimCH-induced [Ca 2+ ] i elevation is inhibited by pre-incubation with α-D-mannoside. FimCH was pre-incubated for 30 minutes with 25 mM α-D-mannoside or D-glucose. PDO7i cells loaded with 5 µM Fluo-4 AM were treated with 10 µg/ml of the pre-incubated FimCH. PDO7i cells were imaged at 488 nm using real-time video fluorescence microscopy and maximal fluorescence after FimCH treatment was subtracted from baseline. (E) Chelating intracellular or extracellular Ca 2+ inhibits elevation of FimCH-induced [Ca 2+ ] i . PD07i cells loaded with 5 µM Fura-2 were pre-incubated with 10 µM BAPTA-AM, 4 mM EGTA, 60 µM 2-APB, 10 µM nifidepine (Ndp), or equivalent amounts of DMSO for 30 minutes, followed by washing and exposure to 10 µg/ml FimCH. Maximal [Ca 2+ ] i was subtracted from baseline [Ca 2+ ] i . Statistical significance is indicated at * p
    Figure Legend Snippet: FimH induces UPIIIa phosphorylation and intracellular calcium elevation. (A) PD07i cells were stimulated with either 10 µg/ml FimCH or 10 µg/ml BSA for 30 minutes, and UPIIIa was immunoprecipitated from 100 µg cell extract with anti-UPIII antibody. Following electrophoresis and blotting, immunoprecipitated proteins were probed with an anti-phosphothreonine antibody (P-Thr). Blots were subsequently re-probed with an anti-UPIII antibody to determine total protein loading (UP3) (B) PD07i cultures were loaded with 5 µM Fura-2 and imaged for 5 minutes at 340 nm and 380 nm using real-time video fluorescence microscopy. FimCH (10 µg/ml) was added after establishing baseline Ca 2+ concentrations collection for 1 min (arrow). The trace is the mean of a representative experiment of three replicates. (C) Extracellular Ca 2+ is required for FimCH-induced urothelial Ca 2+ elevation. PD07i cells were loaded with 5 µM Fura-2 and imaged for 10 minutes in nominal Ca 2+ -free medium. Cells were then exposed to 10 µg/ml FimCH, imaged for 5 minutes followed by addition of Ca 2+ to a final concentration of 2.5 mM to the buffer solution and data were acquired for another 5 min. Baseline Ca 2+ concentration is represented by the dotted line. The trace is the mean of a representative experiment of three replicates. (D) FimCH-induced [Ca 2+ ] i elevation is inhibited by pre-incubation with α-D-mannoside. FimCH was pre-incubated for 30 minutes with 25 mM α-D-mannoside or D-glucose. PDO7i cells loaded with 5 µM Fluo-4 AM were treated with 10 µg/ml of the pre-incubated FimCH. PDO7i cells were imaged at 488 nm using real-time video fluorescence microscopy and maximal fluorescence after FimCH treatment was subtracted from baseline. (E) Chelating intracellular or extracellular Ca 2+ inhibits elevation of FimCH-induced [Ca 2+ ] i . PD07i cells loaded with 5 µM Fura-2 were pre-incubated with 10 µM BAPTA-AM, 4 mM EGTA, 60 µM 2-APB, 10 µM nifidepine (Ndp), or equivalent amounts of DMSO for 30 minutes, followed by washing and exposure to 10 µg/ml FimCH. Maximal [Ca 2+ ] i was subtracted from baseline [Ca 2+ ] i . Statistical significance is indicated at * p

    Techniques Used: Immunoprecipitation, Electrophoresis, Fluorescence, Microscopy, Concentration Assay, Incubation

    26) Product Images from "Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers"

    Article Title: Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

    Journal: Toxins

    doi: 10.3390/toxins10090351

    EspP S263A-stimulated Isc in proximal HCM is Ca 2+ -dependent. ( A ) Representative traces and ( B ) quantitative analysis of BAPTA-AM effect on EspP S263A-stimulated Isc. BAPTAM-AM (25 µM pretreatment for up to 30 min) significantly inhibits EspP S263A-stimulated Isc. ( C ) EspP S263A-stimulated Isc is not inhibited by CaCC inh -A01. ( D ) Esp S263A-stimulated Isc is similar in the buffer containing Cl − (black trace) and without Cl − (red trace). The inset shows that ΔIsc is quantitatively similar in Cl − and Cl − -free buffer; n is the number of monolayers studied.
    Figure Legend Snippet: EspP S263A-stimulated Isc in proximal HCM is Ca 2+ -dependent. ( A ) Representative traces and ( B ) quantitative analysis of BAPTA-AM effect on EspP S263A-stimulated Isc. BAPTAM-AM (25 µM pretreatment for up to 30 min) significantly inhibits EspP S263A-stimulated Isc. ( C ) EspP S263A-stimulated Isc is not inhibited by CaCC inh -A01. ( D ) Esp S263A-stimulated Isc is similar in the buffer containing Cl − (black trace) and without Cl − (red trace). The inset shows that ΔIsc is quantitatively similar in Cl − and Cl − -free buffer; n is the number of monolayers studied.

    Techniques Used: End-sequence Profiling

    27) Product Images from "Pro-inflammatory Ca++-activated K+ channels are inhibited by hydroxychloroquine"

    Article Title: Pro-inflammatory Ca++-activated K+ channels are inhibited by hydroxychloroquine

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01836-8

    HCQ inhibits Ca ++ -activated K + channels. Electrophysiology registers expressed as current density obtained through the whole cell path clamp technique using THP-1 cells. TEA is a K + channel inhibitor, BAPTA AM is an intracellular Ca ++ chelator. Symbols are averaged normalized currents ± SEM (n = 5, p
    Figure Legend Snippet: HCQ inhibits Ca ++ -activated K + channels. Electrophysiology registers expressed as current density obtained through the whole cell path clamp technique using THP-1 cells. TEA is a K + channel inhibitor, BAPTA AM is an intracellular Ca ++ chelator. Symbols are averaged normalized currents ± SEM (n = 5, p

    Techniques Used:

    28) Product Images from "HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase"

    Article Title: HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

    Journal: Herpesviridae

    doi: 10.1186/2042-4280-1-2

    pUS28 Signaling Causes Calcium-Dependent Phosphorylation of Pyk2 at Y402 . (A) FAK-/- fibroblasts were infected with Ad-Trans only or Ad-Trans+Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) Adenovirus-infected FAK-/- were pre-treated for 30 min with BAPTA-AM to chelate intracellular calcium and then stimulated with 40 ng/ml CCL5 for 5 min. Total cell lysates were analyzed via western blot for phospho-Y402 Pyk2 and total Pyk2. For both experiments, blots were stripped and reprobed to verify pUS28-HA expression. Phospho-specific blots were quantified via densitometry using ImageJ software and are expressed as fold change compared to unstimulated, Ad-Trans infected control.
    Figure Legend Snippet: pUS28 Signaling Causes Calcium-Dependent Phosphorylation of Pyk2 at Y402 . (A) FAK-/- fibroblasts were infected with Ad-Trans only or Ad-Trans+Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) Adenovirus-infected FAK-/- were pre-treated for 30 min with BAPTA-AM to chelate intracellular calcium and then stimulated with 40 ng/ml CCL5 for 5 min. Total cell lysates were analyzed via western blot for phospho-Y402 Pyk2 and total Pyk2. For both experiments, blots were stripped and reprobed to verify pUS28-HA expression. Phospho-specific blots were quantified via densitometry using ImageJ software and are expressed as fold change compared to unstimulated, Ad-Trans infected control.

    Techniques Used: Infection, Western Blot, Expressing, Software

    29) Product Images from "Regulation of BDNF-mediated transcription of immediate early gene Arc by intracellular calcium and calmodulin"

    Article Title: Regulation of BDNF-mediated transcription of immediate early gene Arc by intracellular calcium and calmodulin

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.21863

    Activity-dependent ERK phosphorylation does not depend on intracellular calcium. Cortical neurons were stimulated by KCl (50mM) or 5ng/ml BDNF. Pre-treatments with EGTA (2.5mM) or BAPTA-AM (30uM) were applied 30min before stimulation. Samples were collected
    Figure Legend Snippet: Activity-dependent ERK phosphorylation does not depend on intracellular calcium. Cortical neurons were stimulated by KCl (50mM) or 5ng/ml BDNF. Pre-treatments with EGTA (2.5mM) or BAPTA-AM (30uM) were applied 30min before stimulation. Samples were collected

    Techniques Used: Activity Assay

    30) Product Images from "TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response"

    Article Title: TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20150353

    Endothelial ↑[Ca 2+ ] i is required for LBRC targeted recycling. (a–e) Freshly isolated neutrophils (a–d) or monocytes (e) were added to HUVEC monolayers that were pretreated with vehicle (DMSO) or 20 µM BAPTA-AM. LBRC trafficking (a, b, and e), neutrophil adhesion (c), and neutrophil crawling (d) were assessed using our targeted recycling assay, as described in Materials and methods. Representative images (a) demonstrate targeted recycling to the site of neutrophil TEM (bars, 10 µm). Arrows denote the site where LBRC is or should be enriched. Insets in a are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Endothelial ↑[Ca 2+ ] i is required for LBRC targeted recycling. (a–e) Freshly isolated neutrophils (a–d) or monocytes (e) were added to HUVEC monolayers that were pretreated with vehicle (DMSO) or 20 µM BAPTA-AM. LBRC trafficking (a, b, and e), neutrophil adhesion (c), and neutrophil crawling (d) were assessed using our targeted recycling assay, as described in Materials and methods. Representative images (a) demonstrate targeted recycling to the site of neutrophil TEM (bars, 10 µm). Arrows denote the site where LBRC is or should be enriched. Insets in a are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Isolation, Transmission Electron Microscopy

    Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Activation Assay, Transmission Electron Microscopy, Concentration Assay, Fluorescence, Positive Control, Isolation

    Chelation of cytosolic free Ca 2+ in endothelial cells attenuates leukocyte TEM. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM histamine were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 100 total cells were individually quantified for each condition. (b–d) Freshly isolated human neutrophils (b and c) or monocytes (d) in the presence or absence of anti-PECAM were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. (e) Eluate control experiments were performed as previously described ( Mamdouh et al., 2008 ). In brief, BAPTA-AM eluate collected from BAPTA-AM–treated HUVECs was used to resuspend neutrophils, and their ability to transmigrate was subsequently assessed. (b–e) Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Chelation of cytosolic free Ca 2+ in endothelial cells attenuates leukocyte TEM. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM histamine were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 100 total cells were individually quantified for each condition. (b–d) Freshly isolated human neutrophils (b and c) or monocytes (d) in the presence or absence of anti-PECAM were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. (e) Eluate control experiments were performed as previously described ( Mamdouh et al., 2008 ). In brief, BAPTA-AM eluate collected from BAPTA-AM–treated HUVECs was used to resuspend neutrophils, and their ability to transmigrate was subsequently assessed. (b–e) Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Transmission Electron Microscopy, Concentration Assay, Fluorescence, Isolation

    Endothelial ↑[Ca 2+ ] i is required for LBRC targeted recycling. (a–e) Freshly isolated neutrophils (a–d) or monocytes (e) were added to HUVEC monolayers that were pretreated with vehicle (DMSO) or 20 µM BAPTA-AM. LBRC trafficking (a, b, and e), neutrophil adhesion (c), and neutrophil crawling (d) were assessed using our targeted recycling assay, as described in Materials and methods. Representative images (a) demonstrate targeted recycling to the site of neutrophil TEM (bars, 10 µm). Arrows denote the site where LBRC is or should be enriched. Insets in a are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Endothelial ↑[Ca 2+ ] i is required for LBRC targeted recycling. (a–e) Freshly isolated neutrophils (a–d) or monocytes (e) were added to HUVEC monolayers that were pretreated with vehicle (DMSO) or 20 µM BAPTA-AM. LBRC trafficking (a, b, and e), neutrophil adhesion (c), and neutrophil crawling (d) were assessed using our targeted recycling assay, as described in Materials and methods. Representative images (a) demonstrate targeted recycling to the site of neutrophil TEM (bars, 10 µm). Arrows denote the site where LBRC is or should be enriched. Insets in a are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Isolation, Transmission Electron Microscopy

    Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Activation Assay, Transmission Electron Microscopy, Concentration Assay, Fluorescence, Positive Control, Isolation

    Chelation of cytosolic free Ca 2+ in endothelial cells attenuates leukocyte TEM. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM histamine were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 100 total cells were individually quantified for each condition. (b–d) Freshly isolated human neutrophils (b and c) or monocytes (d) in the presence or absence of anti-PECAM were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. (e) Eluate control experiments were performed as previously described ( Mamdouh et al., 2008 ). In brief, BAPTA-AM eluate collected from BAPTA-AM–treated HUVECs was used to resuspend neutrophils, and their ability to transmigrate was subsequently assessed. (b–e) Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P
    Figure Legend Snippet: Chelation of cytosolic free Ca 2+ in endothelial cells attenuates leukocyte TEM. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca 2+ concentration in response to 10 µM histamine were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F 0 ) of three independent experiments. At least 100 total cells were individually quantified for each condition. (b–d) Freshly isolated human neutrophils (b and c) or monocytes (d) in the presence or absence of anti-PECAM were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. (e) Eluate control experiments were performed as previously described ( Mamdouh et al., 2008 ). In brief, BAPTA-AM eluate collected from BAPTA-AM–treated HUVECs was used to resuspend neutrophils, and their ability to transmigrate was subsequently assessed. (b–e) Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P

    Techniques Used: Transmission Electron Microscopy, Concentration Assay, Fluorescence, Isolation

    31) Product Images from "Spatial and Temporal Dynamics of Mitochondrial Membrane Permeability Waves during Apoptosis"

    Article Title: Spatial and Temporal Dynamics of Mitochondrial Membrane Permeability Waves during Apoptosis

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2009.07.056

    Thapsigargin, but not BAPTA, disrupts the wave of cytochrome c release. HeLa cells in 1.2 mM EGTA undergoing TRAIL-mediated apoptosis were treated with 1 mM BAPTA-AM (BAPTA) or 5 μ M thapsigargin. ( A ) Duration of cytochrome c release (95% of all
    Figure Legend Snippet: Thapsigargin, but not BAPTA, disrupts the wave of cytochrome c release. HeLa cells in 1.2 mM EGTA undergoing TRAIL-mediated apoptosis were treated with 1 mM BAPTA-AM (BAPTA) or 5 μ M thapsigargin. ( A ) Duration of cytochrome c release (95% of all

    Techniques Used:

    32) Product Images from "ATP-Induced Increase in Intracellular Calcium Levels and Subsequent Activation of mTOR as Regulators of Skeletal Muscle Hypertrophy"

    Article Title: ATP-Induced Increase in Intracellular Calcium Levels and Subsequent Activation of mTOR as Regulators of Skeletal Muscle Hypertrophy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092804

    ATP induces muscle hypertrophy and alleviates muscle atrophy in an [Ca 2+ ] i - and mTOR-dependent manner. ( A ) Effects of ATP with or without BAPTA-AM or rapamycin on the increase of muscle weight in soleus, plantaris and gastrocnemius muscles ( n = 4). ( B ) The cross-sectional area distributions were plotted as frequency histograms. At least 2800 fibers from 4 different soleus muscles were counted. ( C ) Expression of P2Y 1 and P2Y 2 receptor mRNA in soleus and plantaris muscle ( n = 8). ( D ) Effects of ATP on soleus and gastrocnemius muscles weights in hindlimb-suspended (left) or denervated (right) muscle shown as a percentage of the control group ( n > 4). * p
    Figure Legend Snippet: ATP induces muscle hypertrophy and alleviates muscle atrophy in an [Ca 2+ ] i - and mTOR-dependent manner. ( A ) Effects of ATP with or without BAPTA-AM or rapamycin on the increase of muscle weight in soleus, plantaris and gastrocnemius muscles ( n = 4). ( B ) The cross-sectional area distributions were plotted as frequency histograms. At least 2800 fibers from 4 different soleus muscles were counted. ( C ) Expression of P2Y 1 and P2Y 2 receptor mRNA in soleus and plantaris muscle ( n = 8). ( D ) Effects of ATP on soleus and gastrocnemius muscles weights in hindlimb-suspended (left) or denervated (right) muscle shown as a percentage of the control group ( n > 4). * p

    Techniques Used: Expressing

    ATP upregulates JunB and IL-6 by activating Erk1/2, p38 MAPK and mTOR in C2C12 myotubes. ( A ) Concentration-dependent effects of ATP (left) or UTP (right) on phosphorylation of Erk1/2, p38 MAPK and AMPKα ( n = 4). ( B , C ) Gene expression analysis showing the effects of BAPTA-AM (white), U0126 (blue), SB203580 (yellow) and rapamycin (red) on the ATP-induced expression of JunB ( B ) and IL-6 ( C ) ( n = 4). ( D ) Representative Western blots showing the effects of BAPTA-AM or rapamycin on the ATP-induced increase of JunB ( n = 4). ( E ) Effects of BAPTA-AM or rapamycin on the ATP-induced increase of IL-6 protein level ( n = 4). The concentration of ATP in ( B – E ) was 100 µM and the duration of treatment in all experiments shown in this figure was 30 min. ** p
    Figure Legend Snippet: ATP upregulates JunB and IL-6 by activating Erk1/2, p38 MAPK and mTOR in C2C12 myotubes. ( A ) Concentration-dependent effects of ATP (left) or UTP (right) on phosphorylation of Erk1/2, p38 MAPK and AMPKα ( n = 4). ( B , C ) Gene expression analysis showing the effects of BAPTA-AM (white), U0126 (blue), SB203580 (yellow) and rapamycin (red) on the ATP-induced expression of JunB ( B ) and IL-6 ( C ) ( n = 4). ( D ) Representative Western blots showing the effects of BAPTA-AM or rapamycin on the ATP-induced increase of JunB ( n = 4). ( E ) Effects of BAPTA-AM or rapamycin on the ATP-induced increase of IL-6 protein level ( n = 4). The concentration of ATP in ( B – E ) was 100 µM and the duration of treatment in all experiments shown in this figure was 30 min. ** p

    Techniques Used: Concentration Assay, Expressing, Western Blot

    33) Product Images from "Advanced glycation end products (AGEs) activate mast cells"

    Article Title: Advanced glycation end products (AGEs) activate mast cells

    Journal: British Journal of Pharmacology

    doi: 10.1111/j.1476-5381.2010.00905.x

    Changes in emitted fluorescence (at 510 nm) from Fura-2-loaded mast cells excited alternately at 340 and 380 nm upon stimulation (arrow) with 10 µM Glu-BSA (A) and 10 µM Lact-BSA (B). Traces are representative of three independent experiments. Effect of pretreatment with BAPTA-AM (C) on exocytosis induced by Glu-BSA. Cells were pre-incubated with BAPTA-AM at the indicated concentrations (15 min, 37°C) and then stimulated for 10 min with 1 µM Glu-BSA. Data are mean ± SEM from three independent experiments. Effect of Glu-BSA on mast cell exocytosis in Ca 2+ -free HEPES buffer (D). Cells were stimulated for 10 min at 37°C with 10 µM Glu-BSA. Data are mean ± SEM from four independent experiments. Effects of caffeine (E) and 2-APB (F) on exocytosis induced by Glu-BSA. Cells were pre-incubated with the different agents at the indicated concentrations (15 min, 37°C) and then stimulated for 10 min with 10 µM (E) or 1 µM Glu-BSA (F) at 37°C. Data are mean ± SEM from three independent experiments.
    Figure Legend Snippet: Changes in emitted fluorescence (at 510 nm) from Fura-2-loaded mast cells excited alternately at 340 and 380 nm upon stimulation (arrow) with 10 µM Glu-BSA (A) and 10 µM Lact-BSA (B). Traces are representative of three independent experiments. Effect of pretreatment with BAPTA-AM (C) on exocytosis induced by Glu-BSA. Cells were pre-incubated with BAPTA-AM at the indicated concentrations (15 min, 37°C) and then stimulated for 10 min with 1 µM Glu-BSA. Data are mean ± SEM from three independent experiments. Effect of Glu-BSA on mast cell exocytosis in Ca 2+ -free HEPES buffer (D). Cells were stimulated for 10 min at 37°C with 10 µM Glu-BSA. Data are mean ± SEM from four independent experiments. Effects of caffeine (E) and 2-APB (F) on exocytosis induced by Glu-BSA. Cells were pre-incubated with the different agents at the indicated concentrations (15 min, 37°C) and then stimulated for 10 min with 10 µM (E) or 1 µM Glu-BSA (F) at 37°C. Data are mean ± SEM from three independent experiments.

    Techniques Used: Fluorescence, Incubation

    34) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

    Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.1166-09.2009

    ( a ) Induction of CREB-dependent gene expression by cAMP requires Ca 2+ . Stimulation of 4x CRE luciferase activity by forskolin (10 μM) or 8-Br-cAMP (3 mM) was abolished by adding 3 mM Bapta-free acid (Bapta-FA) or by pre-treating with Bapta-AM (10
    Figure Legend Snippet: ( a ) Induction of CREB-dependent gene expression by cAMP requires Ca 2+ . Stimulation of 4x CRE luciferase activity by forskolin (10 μM) or 8-Br-cAMP (3 mM) was abolished by adding 3 mM Bapta-free acid (Bapta-FA) or by pre-treating with Bapta-AM (10

    Techniques Used: Expressing, Luciferase, Activity Assay

    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
    Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Techniques Used: Plasmid Preparation

    35) Product Images from "Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition"

    Article Title: Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201702187

    RhoA/ROCK pathway activates HDAC6 through a Ca 2+ -dependent mechanism. (A) FRAP analyses for Mito-GFP in distal axons of DRGs cultured on laminin and treated with bath-applied Fc + DMSO, Fc + 3 µM BAPTA-AM, MAG-Fc + DMSO, or MAG-Fc + 3 µM BAPTA-AM are shown as average normalized percentage recovery ± SEM ( n ≥ 9 axons over three culture preparations; **, P ≤ 0.01; ***, P ≤ 0.005 for indicated treatments by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from over 300 to 960 s. (B) End-point FRAP for Mito-GFP in distal axons of DRGs treated with bath-applied Fc vs. MAG-Fc ± 3 µM BAPTA-AM, 10 µM Y27632, 10 µM TubA, or indicated combinations of these inhibitors is shown as average of normalized percentage recovery ± SEM at 960 s after bleach ( n ≥ 9 axons over three culture preparations; ***, P ≤ 0.005 vs. Fc; ## , P ≤ 0.01; ### , P ≤ 0.005 vs. Mag-treated by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from vehicle. (C and D) FRAP for Mito-GFP in distal axons of DRGs treated with RhoA Activator (C) or Thapsigargin (D) is shown as average normalized percentage recovery ± SEM. Data for vehicle control (DMSO), 1 µg/ml of Rho-Activator (+ DMSO), Rho-Activator + 10 µM TubA, 1 µM Thapsigargin (+ DMSO), and Thapsigargin + 10 µM TubA are shown ( n ≥ 9 axons over three culture preparations; *, P ≤ 0.05; ***, P ≤ 0.005 for indicated treatments by two-way ANOVA with Tukey post hoc). (E) Quantification of mitochondrial membrane potential based on red/green fluorescence of JC-1 in axon shafts of DRGs cultured on laminin (Cntl) or aggrecan (CSPG) substrates is shown after treatment with 1 µM Thapsigargin (Thapsi), Thapsi + 10 µM TubA, 1 µg/ml Rho-Activator (Rho-Act’r), or Rho-Act’r + TubA. Values represent average ratio of normalized red/green fluorescent JC-1 signals ± SEM ( n ≥ 20 axons over three culture preparations; **, P ≤ 0.01 vs. control; # , P ≤ 0.05 vs. Thapsi + DMSO; Δ , P ≤ 0.01 vs. Rho-Act’r + DMSO by two-way ANOVA with Tukey post hoc). (F) End-point FRAP for Mito-GFP in distal axons of DRGs cultured on laminin and treated ± 3 µM BAPTA-AM, 1 µg/ml Rho-Act’r, Rho-Act’r + BAPTA-AM is shown as average of normalized percentage recovery ± SEM at 960 s after bleach ( n ≥ 9 axons over three culture preparations; ***, P ≤ 0.005 vs. control; ### , P ≤ 0.005 vs. BAPTA-AM; ΔΔΔ , P ≤ 0.005 vs. Rho-Act’r + DMSO by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from control.
    Figure Legend Snippet: RhoA/ROCK pathway activates HDAC6 through a Ca 2+ -dependent mechanism. (A) FRAP analyses for Mito-GFP in distal axons of DRGs cultured on laminin and treated with bath-applied Fc + DMSO, Fc + 3 µM BAPTA-AM, MAG-Fc + DMSO, or MAG-Fc + 3 µM BAPTA-AM are shown as average normalized percentage recovery ± SEM ( n ≥ 9 axons over three culture preparations; **, P ≤ 0.01; ***, P ≤ 0.005 for indicated treatments by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from over 300 to 960 s. (B) End-point FRAP for Mito-GFP in distal axons of DRGs treated with bath-applied Fc vs. MAG-Fc ± 3 µM BAPTA-AM, 10 µM Y27632, 10 µM TubA, or indicated combinations of these inhibitors is shown as average of normalized percentage recovery ± SEM at 960 s after bleach ( n ≥ 9 axons over three culture preparations; ***, P ≤ 0.005 vs. Fc; ## , P ≤ 0.01; ### , P ≤ 0.005 vs. Mag-treated by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from vehicle. (C and D) FRAP for Mito-GFP in distal axons of DRGs treated with RhoA Activator (C) or Thapsigargin (D) is shown as average normalized percentage recovery ± SEM. Data for vehicle control (DMSO), 1 µg/ml of Rho-Activator (+ DMSO), Rho-Activator + 10 µM TubA, 1 µM Thapsigargin (+ DMSO), and Thapsigargin + 10 µM TubA are shown ( n ≥ 9 axons over three culture preparations; *, P ≤ 0.05; ***, P ≤ 0.005 for indicated treatments by two-way ANOVA with Tukey post hoc). (E) Quantification of mitochondrial membrane potential based on red/green fluorescence of JC-1 in axon shafts of DRGs cultured on laminin (Cntl) or aggrecan (CSPG) substrates is shown after treatment with 1 µM Thapsigargin (Thapsi), Thapsi + 10 µM TubA, 1 µg/ml Rho-Activator (Rho-Act’r), or Rho-Act’r + TubA. Values represent average ratio of normalized red/green fluorescent JC-1 signals ± SEM ( n ≥ 20 axons over three culture preparations; **, P ≤ 0.01 vs. control; # , P ≤ 0.05 vs. Thapsi + DMSO; Δ , P ≤ 0.01 vs. Rho-Act’r + DMSO by two-way ANOVA with Tukey post hoc). (F) End-point FRAP for Mito-GFP in distal axons of DRGs cultured on laminin and treated ± 3 µM BAPTA-AM, 1 µg/ml Rho-Act’r, Rho-Act’r + BAPTA-AM is shown as average of normalized percentage recovery ± SEM at 960 s after bleach ( n ≥ 9 axons over three culture preparations; ***, P ≤ 0.005 vs. control; ### , P ≤ 0.005 vs. BAPTA-AM; ΔΔΔ , P ≤ 0.005 vs. Rho-Act’r + DMSO by two-way ANOVA with Tukey post hoc). BAPTA-AM is not statistically different from control.

    Techniques Used: Cell Culture, Fluorescence, Activated Clotting Time Assay

    36) Product Images from "M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation"

    Article Title: M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00287

    The regulation of KCC2 by m-calpain is independent on [Ca 2+ ] i but is related to KCC2 endocytosis. (A) BAPTA is able to reverse 0 Mg 2+ induced mKCC2 down-regulation, but failed to reverse BDNF induced mKCC2 down-regulation. (B) BAPTA partially reverses the 0 Mg 2+ induced, but not BDNF induced Changes in cytoplasmic KCC2 (cKCC2) down-regulation. (C) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + BAPTA, BDNF and BDNF + BAPTA treated slices. BAPTA intervention partially reverses the 0 Mg 2+ induced, but not BDNF induced the increase of phosphor-serine level of m-calpain. (D–E) Tautomycetin (TMC) can reverse KCC2 down-regulation in plasm membrane (left), but the expression of KCC2 in cytoplasm remained significantly lower than DMSO group (right). In TMC + PD98059 co-treated group the expression of KCC2 is rescued to control level. * p
    Figure Legend Snippet: The regulation of KCC2 by m-calpain is independent on [Ca 2+ ] i but is related to KCC2 endocytosis. (A) BAPTA is able to reverse 0 Mg 2+ induced mKCC2 down-regulation, but failed to reverse BDNF induced mKCC2 down-regulation. (B) BAPTA partially reverses the 0 Mg 2+ induced, but not BDNF induced Changes in cytoplasmic KCC2 (cKCC2) down-regulation. (C) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + BAPTA, BDNF and BDNF + BAPTA treated slices. BAPTA intervention partially reverses the 0 Mg 2+ induced, but not BDNF induced the increase of phosphor-serine level of m-calpain. (D–E) Tautomycetin (TMC) can reverse KCC2 down-regulation in plasm membrane (left), but the expression of KCC2 in cytoplasm remained significantly lower than DMSO group (right). In TMC + PD98059 co-treated group the expression of KCC2 is rescued to control level. * p

    Techniques Used: Expressing

    37) Product Images from "GnRH Regulation of Jun and Atf3 Requires Calcium, Calcineurin, and NFAT"

    Article Title: GnRH Regulation of Jun and Atf3 Requires Calcium, Calcineurin, and NFAT

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2012-1045

    GnRH regulation of IEG accumulation requires calcium. LβT2 cells were serum starved overnight and pretreated with DMSO, BAPTA-AM (50 μ m ), or thapsigargin (1 μ m ) for 1 h. After treatment, cells were then treated with PBS (vehicle)
    Figure Legend Snippet: GnRH regulation of IEG accumulation requires calcium. LβT2 cells were serum starved overnight and pretreated with DMSO, BAPTA-AM (50 μ m ), or thapsigargin (1 μ m ) for 1 h. After treatment, cells were then treated with PBS (vehicle)

    Techniques Used:

    38) Product Images from "Arrest Defective-1 Controls Tumor Cell Behavior by Acetylating Myosin Light Chain Kinase"

    Article Title: Arrest Defective-1 Controls Tumor Cell Behavior by Acetylating Myosin Light Chain Kinase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007451

    hARD1-binding and lysyl-acetylation of MLCK are stimulated by Ca 2+ signaling. (A) The hARD1-MLCK interaction is stimulated by the Ca 2+ signaling. HT1080 cells were co-transfected with 4 µg each of pcARD1 and pcMLCK. The cells were treated with 1 µM ionomycin (IM) and 30 nM phorbol myristate acetate (PMA) for 8 h, or 20 µM BAPTA-AM for 3 h. MLCK was co-immunoprecipitated by anti-ARD1 antibody. Co-precipitated proteins were analyzed by immunoblotting (upper panel). The input levels were analyzed by immunoblotting (lower panel). (B) The lysyl-acetylation of MLCK is augmented by Ca 2+ signaling. Untransfected HT1080 cells were treated with ionomycin and PMA for 8 h or BAPTA-AM for 3 h, alternatively, Trichostatin-A (TSA) at 500 nM was treated for 5 h before cell harvest. The cell lysates were immunoprecipitated with anti-MLCK antibody, and immunoblotted by anti-acetyl-lysine or anti-MLCK antibody. (C) MLCK is acetylated by hARD1. HT1080 cells were transfected with pcARD1 (4 µg) or ARD1-silencing RNA (si-ARD1, 80 nM), and then treated with ionomycin and PMA for 8 h. In the samples immunoprecipitated with anti-MLCK antibody, total MLCK and acetylated MLCK were analyzed by immunoblotting. (D) Endogenous MLCK is acetylated hARD1-dependently. Endogenous MLCK was down-regulated in HT1080 cells using each of three siRNAs (80 nM) targeting different sites of ARD1 mRNA, and then treated with ionomycin and PMA for 8 h. Acetylated MLCK was immunoprecipitated by anti-MLCK antibody and immunoblotted with anti-acetyl-lysine antibody. Input protein levels were analyzed by immuboblotting with specific antibodies.
    Figure Legend Snippet: hARD1-binding and lysyl-acetylation of MLCK are stimulated by Ca 2+ signaling. (A) The hARD1-MLCK interaction is stimulated by the Ca 2+ signaling. HT1080 cells were co-transfected with 4 µg each of pcARD1 and pcMLCK. The cells were treated with 1 µM ionomycin (IM) and 30 nM phorbol myristate acetate (PMA) for 8 h, or 20 µM BAPTA-AM for 3 h. MLCK was co-immunoprecipitated by anti-ARD1 antibody. Co-precipitated proteins were analyzed by immunoblotting (upper panel). The input levels were analyzed by immunoblotting (lower panel). (B) The lysyl-acetylation of MLCK is augmented by Ca 2+ signaling. Untransfected HT1080 cells were treated with ionomycin and PMA for 8 h or BAPTA-AM for 3 h, alternatively, Trichostatin-A (TSA) at 500 nM was treated for 5 h before cell harvest. The cell lysates were immunoprecipitated with anti-MLCK antibody, and immunoblotted by anti-acetyl-lysine or anti-MLCK antibody. (C) MLCK is acetylated by hARD1. HT1080 cells were transfected with pcARD1 (4 µg) or ARD1-silencing RNA (si-ARD1, 80 nM), and then treated with ionomycin and PMA for 8 h. In the samples immunoprecipitated with anti-MLCK antibody, total MLCK and acetylated MLCK were analyzed by immunoblotting. (D) Endogenous MLCK is acetylated hARD1-dependently. Endogenous MLCK was down-regulated in HT1080 cells using each of three siRNAs (80 nM) targeting different sites of ARD1 mRNA, and then treated with ionomycin and PMA for 8 h. Acetylated MLCK was immunoprecipitated by anti-MLCK antibody and immunoblotted with anti-acetyl-lysine antibody. Input protein levels were analyzed by immuboblotting with specific antibodies.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation

    39) Product Images from "CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy"

    Article Title: CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy

    Journal: Diabetes

    doi: 10.2337/db16-0051

    CD40 ligation in Müller cells causes Tyr783 phosphorylation (p) of PLCγ1 and PLC-dependent secretion of ATP. A : CD40 + Müller cells were incubated with or without BAPTA-AM, followed by stimulation with CD154 and measurement of extracellular ATP. Concentrations of extracellular ATP at time 0 and at 15 min of incubation are shown. B : CD40 + human Müller cells were incubated with CD154. Expression of p-Tyr783 PLCγ1 and total PLCγ1 were assessed by immunoblot. The bars represent quantification of relative p-Tyr783 PLCγ1 from three different experiments. C : CD40 + Müller cells were incubated with U73122 or U73343, followed by stimulation with CD154 and measurement of extracellular ATP. Concentrations of extracellular ATP at time 0 and at 15 min of incubation are shown. Results are presented as mean ± SEM ( n = 3). ** P
    Figure Legend Snippet: CD40 ligation in Müller cells causes Tyr783 phosphorylation (p) of PLCγ1 and PLC-dependent secretion of ATP. A : CD40 + Müller cells were incubated with or without BAPTA-AM, followed by stimulation with CD154 and measurement of extracellular ATP. Concentrations of extracellular ATP at time 0 and at 15 min of incubation are shown. B : CD40 + human Müller cells were incubated with CD154. Expression of p-Tyr783 PLCγ1 and total PLCγ1 were assessed by immunoblot. The bars represent quantification of relative p-Tyr783 PLCγ1 from three different experiments. C : CD40 + Müller cells were incubated with U73122 or U73343, followed by stimulation with CD154 and measurement of extracellular ATP. Concentrations of extracellular ATP at time 0 and at 15 min of incubation are shown. Results are presented as mean ± SEM ( n = 3). ** P

    Techniques Used: Ligation, Planar Chromatography, Incubation, Expressing

    40) Product Images from "Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages"

    Article Title: Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20060943

    Role of exocytosis in TLR4 translocation to the plasma membrane after oxidant stress. Raw 264.7 cells remained untreated (control) or exposed to DMSO vehicle or 1 μM jaspakinolide (JAS) (A and C) for 30 min or 10 μM BAPTA/AM for 10 min (B and D). Cells were then exposed to 100 μM H 2 O 2 for1 h or 0.1 μg/ml LPS for 30 min, followed by staining live with anti–CD11b-FITC (A and B) or anti-TLR4/MD2-FITC at 4°C (C and D). Fluorescence was analyzed by flow cytometry. Data are mean ± SEM of n = 4 per group. *, P
    Figure Legend Snippet: Role of exocytosis in TLR4 translocation to the plasma membrane after oxidant stress. Raw 264.7 cells remained untreated (control) or exposed to DMSO vehicle or 1 μM jaspakinolide (JAS) (A and C) for 30 min or 10 μM BAPTA/AM for 10 min (B and D). Cells were then exposed to 100 μM H 2 O 2 for1 h or 0.1 μg/ml LPS for 30 min, followed by staining live with anti–CD11b-FITC (A and B) or anti-TLR4/MD2-FITC at 4°C (C and D). Fluorescence was analyzed by flow cytometry. Data are mean ± SEM of n = 4 per group. *, P

    Techniques Used: Translocation Assay, Staining, Fluorescence, Flow Cytometry, Cytometry

    Related Articles

    Isolation:

    Article Title: The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes
    Article Snippet: .. Drug treatments P. falciparum merozoites were isolated in IC buffer and treated for 15 mins with the following at concentrations shown: 100 µM IBMX (3-isobutyl-1-methylxanthine, Calbiochem, USA), 50 µM KH7 (Calbiochem, USA), 100 µM ACTZ (acetazolamide, Sigma, USA), 20 µM DiB-cAMP (adenosine 3,5 cyclic monophosphate, N6 , O2 - dibutyryl sodium salt, Calbiochem, USA), 10 µM Ca2+ ionophore A23187 (Calbiochem, USA), 50 µM BAPTA-AM (Calbiochem, USA), 10 µM U73122 (Calbiochem, USA), 10 µM U73343 (Calbiochem, USA), 200 µM Epac agonist (8-pCPT-2’-O-Me-cAMP, Sigma, USA), 25 µM Epac antagonist ESI-05 (Biolog, Germany), 25 µM Epac antagonist ESI-09 (Biolog, Germany) and 50 µM geranyl geranyl transferase inhibitor, GGTI 298, which disrupts Rap1 function. ..

    other:

    Article Title: Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation
    Article Snippet: Reagents ABT-737 was purchased by Selleckem (Souffelweyersheim, France) and BAPTA-AM was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

    Glo Assay:

    Article Title: Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells
    Article Snippet: .. FBS was obtained from Connectorate (Dietikon, Switzerland), Cell Titer Glo® assay kit from Promega (Madison, WI, USA) and thapsigargin, AICAR, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), 6-methyl-2-(phenylethynyl)-pyridine (MPEP), BAPTA-AM, pertussis toxin (PTX), dantrolene sodium and sodium phenylbutyrate (PBA) from Sigma-Aldrich (St. Louis, MO, USA). .. Compound C and xestospongin C were obtained from Calbiochem (San Diego, CA, USA).

    Activation Assay:

    Article Title: Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue
    Article Snippet: .. To characterize the mechanisms involved in platelet activation by cell free histone H2A, platelets were pretreated with the calcium chelator BAPTA-AM (Sigma) (20 μM) or anti-TLR4 neutralizing antibodies (eBioscience 169917–82) (20 μg/mL) for 30 min prior stimulation with histone H2A. .. Flow cytometry analysis Platelets (1–5 x 106 ) were incubated with FITC-conjugated anti-CD41 (BD Phamingen, CA) (1:20), PE-conjugated anti-CD62-P (BD Pharmingen, CA) (1:20) and APC-conjugated anti-HLA-A, B, C (Biolegend, CA) (1:50) for 30 min at 37°C.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore bapta am
    Crosstalk between cAMP and Ca 2+ in P. falciparum merozoites. A) Ca 2+ does not regulate cytosolic cAMP levels. P. falciparum merozoites were transferred from IC to EC buffer with or without treatment with <t>BAPTA-AM</t> or <t>U73122.</t> Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca 2+ is inhibited by ACβ inhibitor KH7. P. falciparum merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca 2+ levels in P. falciparum merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca 2+ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca 2+ . P. falciparum PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca 2+ levels rise normally following transfer of P. falciparum merozoites from IC to EC buffer. Cytosolic Ca 2+ levels do not rise when P. falciparum PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca 2+ levels in merozoites. D) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca 2+ stimulated by Epac agonist. E) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca 2+ stimulated by Epac agonist. F) Cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca 2+ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca 2+ .
    Bapta Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bapta am/product/Millipore
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    bapta am - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Crosstalk between cAMP and Ca 2+ in P. falciparum merozoites. A) Ca 2+ does not regulate cytosolic cAMP levels. P. falciparum merozoites were transferred from IC to EC buffer with or without treatment with BAPTA-AM or U73122. Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca 2+ is inhibited by ACβ inhibitor KH7. P. falciparum merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca 2+ levels in P. falciparum merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca 2+ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca 2+ . P. falciparum PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca 2+ levels rise normally following transfer of P. falciparum merozoites from IC to EC buffer. Cytosolic Ca 2+ levels do not rise when P. falciparum PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca 2+ levels in merozoites. D) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca 2+ stimulated by Epac agonist. E) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca 2+ stimulated by Epac agonist. F) Cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca 2+ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca 2+ .

    Journal: PLoS Pathogens

    Article Title: The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

    doi: 10.1371/journal.ppat.1004520

    Figure Lengend Snippet: Crosstalk between cAMP and Ca 2+ in P. falciparum merozoites. A) Ca 2+ does not regulate cytosolic cAMP levels. P. falciparum merozoites were transferred from IC to EC buffer with or without treatment with BAPTA-AM or U73122. Levels of cytosolic cAMP were measured in merozoite lysates before and after transfer to EC buffer. Fold changes in cAMP levels per µg of merozoite protein (mean ± SD from 3 independent experiments) in different conditions relative to cAMP levels in IC buffer (mean ± SD) from 3 independent experiments are reported. Treatment of merozoites with BAPTA-AM or U73122 does not have any effect on rise in intracellular cAMP levels following transfer from IC to EC buffer. B) Rise in cytosolic Ca 2+ is inhibited by ACβ inhibitor KH7. P. falciparum merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer with or without treatment with KH7. Cytosolic Ca 2+ levels in P. falciparum merozoites were measured before and after transfer by flow cytometry. Treatment with KH7 inhibits the rise in cytosolic Ca 2+ following transfer to EC buffer. C) PKA does not regulate cytosolic Ca 2+ . P. falciparum PHL dhfr-PfPKAr merozoites were loaded with Fluo-4AM and transferred from IC to EC buffer or from IC to IC+DiB or from IC to EC+DiB. Cytosolic Ca 2+ levels rise normally following transfer of P. falciparum merozoites from IC to EC buffer. Cytosolic Ca 2+ levels do not rise when P. falciparum PHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca 2+ levels in merozoites. D) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer or from IC to IC buffer containing Epac agonist 8-Pcpt-2’-O-Me-cAMP (IC+Epac agonist), DiB (IC+DiB), or Epac agonist and Epac inhibitors (IC+Epac agonist+ESI-09 or IC+Epac agonist+ESI-05). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or IC buffer to IC+Epac agonist, but not when they are transferred from IC buffer to IC+DiB. EPAC1 antagonist ESI-09 inhibits rise in Ca 2+ stimulated by Epac agonist. E) Regulation of cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites by Epac agonist, PLC inhibitor and Rap1 inhibitor. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer, or from IC to IC buffer containing Epac agonist (IC+Epac agonist), or IC buffer containing Epac agonist and PLC inhibitor (IC+Epac agonist+U73122), or IC buffer containing Epac agonist and Rap1 inhibitor GGTI298 (IC+Epac agonist+GGTI298). Cytosolic Ca 2+ levels rise when merozoites are transferred from IC to EC buffer, or from IC to IC+Epac agonist. PLC inhibitor U73122 and Rap1 inhibitor GGTI298 inhibit rise in cytosolic Ca 2+ stimulated by Epac agonist. F) Cytosolic Ca 2+ levels in P. falciparum 3D7 merozoites following transfer to EC buffer in presence of Epac and Rap1 inhibitors. P falciparum merozoites loaded with Fluo-4AM were transferred from IC to EC buffer containing Epac inhibitors (EC+ESI-09 or EC+ESI-05), or EC buffer containing Rap1 inhibitor (EC+GGTI298). Cytosolic Ca 2+ rises when merozoites are transferred from IC to EC buffer. Presence of Epac inhibitor ESI-09 and Rap1 inhibitor GGTI298 inhibits rise in cytosolic Ca 2+ .

    Article Snippet: Drug treatments P. falciparum merozoites were isolated in IC buffer and treated for 15 mins with the following at concentrations shown: 100 µM IBMX (3-isobutyl-1-methylxanthine, Calbiochem, USA), 50 µM KH7 (Calbiochem, USA), 100 µM ACTZ (acetazolamide, Sigma, USA), 20 µM DiB-cAMP (adenosine 3,5 cyclic monophosphate, N6 , O2 - dibutyryl sodium salt, Calbiochem, USA), 10 µM Ca2+ ionophore A23187 (Calbiochem, USA), 50 µM BAPTA-AM (Calbiochem, USA), 10 µM U73122 (Calbiochem, USA), 10 µM U73343 (Calbiochem, USA), 200 µM Epac agonist (8-pCPT-2’-O-Me-cAMP, Sigma, USA), 25 µM Epac antagonist ESI-05 (Biolog, Germany), 25 µM Epac antagonist ESI-09 (Biolog, Germany) and 50 µM geranyl geranyl transferase inhibitor, GGTI 298, which disrupts Rap1 function.

    Techniques: Flow Cytometry, Cytometry, Planar Chromatography

    N -desmethyldauricine (LP-4) induces autophagy via CaMKK-β -AMPK -mTOR signaling pathways. (A) LP-4 activated the AMPK-mTOR cascade. Cells treated with LP-4 (10 μM) or positive control (rapamycin, 300 nM) for 24 h was analyzed for the expression of p-p70S6K, total p70S6K, p-AMPK, AMPK, and actin by Western blot. (B) Inhibitors for AMPK, CaMKK-β and calcium chelator inhibited autophagy induction by LP-4. EGFP-LC3 transfected HeLa cells were treated with DMSO (Ctrl) or LP-4 (10 μM) in the presence or absence of AMPK inhibitor compound C (CC, 10 μM), CaMKK β inhibitor (STO-609, 25 μM) or calcium chelator, BAPTA/AM (BM, 10 μM) for 4 h. The cells were then fixed for fluorescence microscopic analysis and quantitation of autophagic cells. (C) Compound C, STO-609 and BAPTA/AM inhibited the conversion of LC3-II in HeLa cells. Cells treated with LP-4 (10 μM) with or without the presence of compound C (CC, 10 μM), STO-609 (25 μM) or BAPTA/AM (10 μM) for 4 h were analyzed for the protein expression of LC3-II (LC3-I, 18 kDa; LC3-II, 16 kDa). (D) Calcium chelator (BAPTA/AM) suppressed the cell death induced by LP-4 in HeLa. Cells treated with LP-4 (10 μM) with the presence of BAPTA/AM (BM, 2.5 μM) for 24 h were subjected to flow cytometry analysis after annexin V staining. Bar chart indicated the percentage of apoptotic cells after treatments. Data were the means of three independent experiments; error bars, SD ∗∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization

    doi: 10.3389/fphar.2017.00388

    Figure Lengend Snippet: N -desmethyldauricine (LP-4) induces autophagy via CaMKK-β -AMPK -mTOR signaling pathways. (A) LP-4 activated the AMPK-mTOR cascade. Cells treated with LP-4 (10 μM) or positive control (rapamycin, 300 nM) for 24 h was analyzed for the expression of p-p70S6K, total p70S6K, p-AMPK, AMPK, and actin by Western blot. (B) Inhibitors for AMPK, CaMKK-β and calcium chelator inhibited autophagy induction by LP-4. EGFP-LC3 transfected HeLa cells were treated with DMSO (Ctrl) or LP-4 (10 μM) in the presence or absence of AMPK inhibitor compound C (CC, 10 μM), CaMKK β inhibitor (STO-609, 25 μM) or calcium chelator, BAPTA/AM (BM, 10 μM) for 4 h. The cells were then fixed for fluorescence microscopic analysis and quantitation of autophagic cells. (C) Compound C, STO-609 and BAPTA/AM inhibited the conversion of LC3-II in HeLa cells. Cells treated with LP-4 (10 μM) with or without the presence of compound C (CC, 10 μM), STO-609 (25 μM) or BAPTA/AM (10 μM) for 4 h were analyzed for the protein expression of LC3-II (LC3-I, 18 kDa; LC3-II, 16 kDa). (D) Calcium chelator (BAPTA/AM) suppressed the cell death induced by LP-4 in HeLa. Cells treated with LP-4 (10 μM) with the presence of BAPTA/AM (BM, 2.5 μM) for 24 h were subjected to flow cytometry analysis after annexin V staining. Bar chart indicated the percentage of apoptotic cells after treatments. Data were the means of three independent experiments; error bars, SD ∗∗∗ P

    Article Snippet: BAPTA/AM (BM), compound C (CC), E64D, pepstatin A, thapsigargin, inositol trisphosphate, STO-609 and AMD3100 were purchased from Calbiochem (San Diego, CA, United States).

    Techniques: Positive Control, Expressing, Western Blot, Transfection, Fluorescence, Quantitation Assay, Flow Cytometry, Cytometry, Staining

    N -desmethyldauricine (LP-4) induces autophagic cell death in apoptosis resistant cells. (A) Cell death measurement (%) in Atg7- wild-type and -deficient MEFs after LP-4 treatment. (B) Cytotoxicity (IC 50 value) of LP-4 in caspase WT/-3/-7/-8, caspase -3 and -7 DKO, Bax-Bak DKO -wild-type and -deficient MEFs. These panel of MEFs were incubated with LP-4 (0.19–100 μM) for 3 days. MTT assay were performed to evaluate the mean IC 50 of LP-4. (C) BAPTA/AM inhibited the autophagic effect of LP-4 in apoptosis-resistant cells. Bax-Bak DKO deficient MEFs were treated with 10 μM of LP-4 in the presence of calcium chelator, BAPTA/AM (BM, 10 μM), for 4 h. The cells were then fixed and incubated with anti-LC3B and TRITC-conjugated secondary antibodies before fluorescence microscopic analysis. (D,E) Live-cell imaging of LP-4-induced autophagic cell death. Cells transfected with EGFP-LC3 were treated with 10 μM of LP-4. Cells were then placed inside the 37°C imaging chamber supplied with 5% of CO 2 for monitoring of autophagy and autophagic cell death. Live-cell fluorescent and bright field images were captured for 4 h with 10 min time-intervals. Yellow arrows indicated autophagy induction with EGFP-LC3 accumulation; Red arrows indicated autophagic cell death. ∗∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization

    doi: 10.3389/fphar.2017.00388

    Figure Lengend Snippet: N -desmethyldauricine (LP-4) induces autophagic cell death in apoptosis resistant cells. (A) Cell death measurement (%) in Atg7- wild-type and -deficient MEFs after LP-4 treatment. (B) Cytotoxicity (IC 50 value) of LP-4 in caspase WT/-3/-7/-8, caspase -3 and -7 DKO, Bax-Bak DKO -wild-type and -deficient MEFs. These panel of MEFs were incubated with LP-4 (0.19–100 μM) for 3 days. MTT assay were performed to evaluate the mean IC 50 of LP-4. (C) BAPTA/AM inhibited the autophagic effect of LP-4 in apoptosis-resistant cells. Bax-Bak DKO deficient MEFs were treated with 10 μM of LP-4 in the presence of calcium chelator, BAPTA/AM (BM, 10 μM), for 4 h. The cells were then fixed and incubated with anti-LC3B and TRITC-conjugated secondary antibodies before fluorescence microscopic analysis. (D,E) Live-cell imaging of LP-4-induced autophagic cell death. Cells transfected with EGFP-LC3 were treated with 10 μM of LP-4. Cells were then placed inside the 37°C imaging chamber supplied with 5% of CO 2 for monitoring of autophagy and autophagic cell death. Live-cell fluorescent and bright field images were captured for 4 h with 10 min time-intervals. Yellow arrows indicated autophagy induction with EGFP-LC3 accumulation; Red arrows indicated autophagic cell death. ∗∗∗ P

    Article Snippet: BAPTA/AM (BM), compound C (CC), E64D, pepstatin A, thapsigargin, inositol trisphosphate, STO-609 and AMD3100 were purchased from Calbiochem (San Diego, CA, United States).

    Techniques: Incubation, MTT Assay, Fluorescence, Live Cell Imaging, Transfection, Imaging

    N -desmethyldauricine (LP-4) induces autophagic cell death in apoptosis resistant cancer cells. (A) BAPTA/AM inhibited cell death induced by LP-4 in apoptosis-resistant cancer cells. DLD-1 Bax-Bak DKO colon cancer cells treated with LP-4 (30 μM) in the presence of 10 μM of BAPTA/AM for 24 h were assayed by flow cytometry after annexin V staining. (B) BAPTA/AM abolished the LP-4-induced calcium dynamic change in apoptosis-resistant cancer cells. DLD-1 Bax-Bak DKO colon cancer cells stained with FLIPR Calcium 6 Assay Kit were treated with 30 μM of LP-4, with or without pretreatment of BAPTA/AM (10 μM), then immediately subjected to calcium dynamic measurement by FLIPR Tetra High-Throughput Cellular Screening System. Data from the chart represents mean values ± SD of three independent experiments. (C) Drug-resistant effects of LP-4 on p53-deficient apoptosis-resistant cancer cells. HCT-116 p53 wild-type and deficient colon cancer cells were incubated with different concentrations of LP-4, taxol and etoposide for 3 days. Cytotoxicity of these compounds were measured by MTT assay and shown as the mean IC 50 . (D) BAPTA/AM recovered the cell death from LP-4 treatment in p53-deficient apoptosis-resistant cancer cells. HCT-116 p53-deficient colon cancer cells treated with LP-4 (10 μM) in the presence of 10 μM of BAPTA/AM for 24 h were assayed by flow cytometry after annexin V staining. ∗∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization

    doi: 10.3389/fphar.2017.00388

    Figure Lengend Snippet: N -desmethyldauricine (LP-4) induces autophagic cell death in apoptosis resistant cancer cells. (A) BAPTA/AM inhibited cell death induced by LP-4 in apoptosis-resistant cancer cells. DLD-1 Bax-Bak DKO colon cancer cells treated with LP-4 (30 μM) in the presence of 10 μM of BAPTA/AM for 24 h were assayed by flow cytometry after annexin V staining. (B) BAPTA/AM abolished the LP-4-induced calcium dynamic change in apoptosis-resistant cancer cells. DLD-1 Bax-Bak DKO colon cancer cells stained with FLIPR Calcium 6 Assay Kit were treated with 30 μM of LP-4, with or without pretreatment of BAPTA/AM (10 μM), then immediately subjected to calcium dynamic measurement by FLIPR Tetra High-Throughput Cellular Screening System. Data from the chart represents mean values ± SD of three independent experiments. (C) Drug-resistant effects of LP-4 on p53-deficient apoptosis-resistant cancer cells. HCT-116 p53 wild-type and deficient colon cancer cells were incubated with different concentrations of LP-4, taxol and etoposide for 3 days. Cytotoxicity of these compounds were measured by MTT assay and shown as the mean IC 50 . (D) BAPTA/AM recovered the cell death from LP-4 treatment in p53-deficient apoptosis-resistant cancer cells. HCT-116 p53-deficient colon cancer cells treated with LP-4 (10 μM) in the presence of 10 μM of BAPTA/AM for 24 h were assayed by flow cytometry after annexin V staining. ∗∗∗ P

    Article Snippet: BAPTA/AM (BM), compound C (CC), E64D, pepstatin A, thapsigargin, inositol trisphosphate, STO-609 and AMD3100 were purchased from Calbiochem (San Diego, CA, United States).

    Techniques: Flow Cytometry, Cytometry, Staining, High Throughput Screening Assay, Incubation, MTT Assay

    Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P

    Journal: American Journal of Physiology. Renal Physiology

    Article Title: Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

    doi: 10.1152/ajprenal.00108.2008

    Figure Lengend Snippet: Top : time course of changes in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP in MDCK control cells ( left ) and in cells preloaded for 30 min with 25 μM BAPTA-AM ( right ). Extracellular solution contained 2 mM Ca 2+ . Bottom : ATP produced no significant change in the fluorescence ratio in BAPTA-loaded cells (means ± SE of 30 cells). *Significantly different ( P

    Article Snippet: When MDCK cells were preloaded with BAPTA-AM to chelate intracellular calcium ( , ), there was no significant increase in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP, as expected, ( , top ) and the inhibitory actions of both ATP and adenosine on BGT1 transport were blocked completely ( , bottom ).

    Techniques: Fluorescence, Produced

    Distribution of BGT1 protein (green) in MDCK cell monolayers is primarily in the plasma membrane after hypertonic stress (500 mosmol/kgH 2 O) for 24 h ( B ) in marked contrast to cells maintained in isotonic medium ( A ). Treatment of hypertonic cells for 30 min with 0.1 mM ATP ( C ) or 0.1 mM adenosine ( D ) produced punctuate green fluorescence (arrows) in the cytoplasm of many cells, consistent with endocytic internalization of BGT1 protein. In contrast, almost no intracellular punctuate green fluorescence was observed when BAPTA-loaded cells were treated with 0.1 mM ATP ( F ), compared with the action of ATP on control cells ( E ). Cells in A–D were fixed in paraformaldehyde, permeabilized, and BGT1 was detected with BGT1 antibody from Proteintech. Cells in E–F were methanol fixed and BGT1 was detected with antibody provided by Dr. H. M. Kwon. Nucleic acids and nuclei were counterstained with propidium iodide (red). Bar = 20 μm.

    Journal: American Journal of Physiology. Renal Physiology

    Article Title: Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

    doi: 10.1152/ajprenal.00108.2008

    Figure Lengend Snippet: Distribution of BGT1 protein (green) in MDCK cell monolayers is primarily in the plasma membrane after hypertonic stress (500 mosmol/kgH 2 O) for 24 h ( B ) in marked contrast to cells maintained in isotonic medium ( A ). Treatment of hypertonic cells for 30 min with 0.1 mM ATP ( C ) or 0.1 mM adenosine ( D ) produced punctuate green fluorescence (arrows) in the cytoplasm of many cells, consistent with endocytic internalization of BGT1 protein. In contrast, almost no intracellular punctuate green fluorescence was observed when BAPTA-loaded cells were treated with 0.1 mM ATP ( F ), compared with the action of ATP on control cells ( E ). Cells in A–D were fixed in paraformaldehyde, permeabilized, and BGT1 was detected with BGT1 antibody from Proteintech. Cells in E–F were methanol fixed and BGT1 was detected with antibody provided by Dr. H. M. Kwon. Nucleic acids and nuclei were counterstained with propidium iodide (red). Bar = 20 μm.

    Article Snippet: When MDCK cells were preloaded with BAPTA-AM to chelate intracellular calcium ( , ), there was no significant increase in the fura 2 fluorescence ratio in response to addition of 0.1 mM ATP, as expected, ( , top ) and the inhibitory actions of both ATP and adenosine on BGT1 transport were blocked completely ( , bottom ).

    Techniques: Produced, Fluorescence

    Importance of intracellular signaling cascades in the pMV-absorption. (A) Wild type (WT), CD28 −/− , and LFA-1 −/− 2C T cells, pre- treated with the respective mAbs, were cultured with or without the peptide-loaded L d B7-1ICAM-1 pMVs, as indicated, followed by mAb staining for B7-1 and flow cytometric analysis. Mean fluorescence intensities (MFIs) of B7-1 staining relative to that of WT 2C T cells, pre-treated with control mAb and cultured with the QL9-loaded pMVs, were plotted. Note that P1A peptide forms complex with L d but L d /P1A complex is not recognized by 2C TCR [8] . (B) WT 2C T cells, pre-treated with the respective mAbs, were cultured with the peptide-loaded pMVs for different periods of time, as indicated, and stained for B7-1. (C) WT 2C T cells were treated with PP2 (PP), Wortmannin (WM), Pervanadate (PV), Cytochalasin D (CT), BAPTA/AM (BA), Forskolin (FS), Cyclosporin A (CC) and Nocodazole (NZ), respectively, as indicated, before culture with the QL9-loaded pMVs. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted.

    Journal: PLoS ONE

    Article Title: A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation

    doi: 10.1371/journal.pone.0006044

    Figure Lengend Snippet: Importance of intracellular signaling cascades in the pMV-absorption. (A) Wild type (WT), CD28 −/− , and LFA-1 −/− 2C T cells, pre- treated with the respective mAbs, were cultured with or without the peptide-loaded L d B7-1ICAM-1 pMVs, as indicated, followed by mAb staining for B7-1 and flow cytometric analysis. Mean fluorescence intensities (MFIs) of B7-1 staining relative to that of WT 2C T cells, pre-treated with control mAb and cultured with the QL9-loaded pMVs, were plotted. Note that P1A peptide forms complex with L d but L d /P1A complex is not recognized by 2C TCR [8] . (B) WT 2C T cells, pre-treated with the respective mAbs, were cultured with the peptide-loaded pMVs for different periods of time, as indicated, and stained for B7-1. (C) WT 2C T cells were treated with PP2 (PP), Wortmannin (WM), Pervanadate (PV), Cytochalasin D (CT), BAPTA/AM (BA), Forskolin (FS), Cyclosporin A (CC) and Nocodazole (NZ), respectively, as indicated, before culture with the QL9-loaded pMVs. MFIs of the B7-1 staining relative to that of 2C T cells treated with DMSO alone were plotted.

    Article Snippet: PP2, Wortmannin, Cyclosporin A, Cytochalasin D, Nocodazole, BAPTA/AM, and Forskolin were purchased from EMD Bioscience.

    Techniques: Cell Culture, Staining, Flow Cytometry, Fluorescence