thapsigargin  (Tocris)

 
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  • 99
    Name:
    Thapsigargin
    Description:
    Potent inhibitor of SERCA ATPase
    Catalog Number:
    1138
    Price:
    None
    Purity:
    ≥97% (HPLC)
    Category:
    Ca2 ATPase Inhibitors Ca2 ATPase ATPases Enzymes Pharmacology
    Formula:
    (3S,3aR,4S,6S,6AR,7S,8S,9bS)-6-(Acetyloxy)-2,3,3a,4,5,6,6a,7,8,9b-decahydro-3,3a-dihydroxy-3,6,9-trimethyl-8-[[(2Z)-2-methyl-1-oxo-2-butenyl]oxy]-2-oxo-4-(1-oxobutoxy)azuleno[4,5-b]furan-7-yl octanoate
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    Structured Review

    Tocris thapsigargin
    Thapsigargin
    Potent inhibitor of SERCA ATPase
    https://www.bioz.com/result/thapsigargin/product/Tocris
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Distinct roles of ?- and ?CaMKII in controlling long-term potentiation of GABAA-receptor mediated transmission in murine Purkinje cells"

    Article Title: Distinct roles of ?- and ?CaMKII in controlling long-term potentiation of GABAA-receptor mediated transmission in murine Purkinje cells

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00016

    GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with thapsigargin abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.
    Figure Legend Snippet: GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with thapsigargin abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.

    Techniques Used: Inhibition, Activation Assay, Knock-Out, Mouse Assay

    2) Product Images from "Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes"

    Article Title: Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes

    Journal: Experimental Neurobiology

    doi: 10.5607/en.2017.26.1.42

    Astrocytic SOCE is mediated by Orai channels in combination with Stim1. (a) Schematic illustration of astrocyte Ca 2+ imaging in inverted microscope using ratiometric Ca 2+ dye, Fura-2 AM. (b) Experimental protocol of Ca 2+ imaging. Ca 2+ free HEPES solution containing 200 nM EGTA was bath applied for 1200 s. Meanwhile, 1 µM thapsigargin, the SERCA inhibitor, was applied for 120 s after 60 s baseline to induce Ca 2+ release from ER. External solution was changed to the normal HEPES solution containing 2 mM Ca 2+ at 1200 s. (c) Left top, Fura-2 AM loaded cells. Left bottom, Orai1, Orai2, and Orai3 shRNA co-transfected cells expressing mCherry or EGFP. Right top, representative Ca 2+ imaging 340/380 ratio images taken at 1 min, 3 min, 20 min, 25 min. Right bottom, representative 340/380 ratio traces of two cells. Orai1/2/3 shRNA transfected cell is indicated with red mark and endogenous control was indicated with yellow mark. (d~j) Average of total Ca 2+ imaging traces and scatter plots of ER Ca 2+ release (Release), store operated Ca 2+ entry (Entry) and Entry/Release ratio (Ratio) of primary cultured astrocytes transfected with scrambled shRNA (d), Orai1 shRNA (e), Orai2 shRNA (f), Orai3 shRNA (g), TrpC1 shRNA (h), Orai1/2/3 shRNA (i), Stim1 shRNA (j). Scale bar indicates 0.5 for y axis and 100s for x axis. Tg: thapsigargin.
    Figure Legend Snippet: Astrocytic SOCE is mediated by Orai channels in combination with Stim1. (a) Schematic illustration of astrocyte Ca 2+ imaging in inverted microscope using ratiometric Ca 2+ dye, Fura-2 AM. (b) Experimental protocol of Ca 2+ imaging. Ca 2+ free HEPES solution containing 200 nM EGTA was bath applied for 1200 s. Meanwhile, 1 µM thapsigargin, the SERCA inhibitor, was applied for 120 s after 60 s baseline to induce Ca 2+ release from ER. External solution was changed to the normal HEPES solution containing 2 mM Ca 2+ at 1200 s. (c) Left top, Fura-2 AM loaded cells. Left bottom, Orai1, Orai2, and Orai3 shRNA co-transfected cells expressing mCherry or EGFP. Right top, representative Ca 2+ imaging 340/380 ratio images taken at 1 min, 3 min, 20 min, 25 min. Right bottom, representative 340/380 ratio traces of two cells. Orai1/2/3 shRNA transfected cell is indicated with red mark and endogenous control was indicated with yellow mark. (d~j) Average of total Ca 2+ imaging traces and scatter plots of ER Ca 2+ release (Release), store operated Ca 2+ entry (Entry) and Entry/Release ratio (Ratio) of primary cultured astrocytes transfected with scrambled shRNA (d), Orai1 shRNA (e), Orai2 shRNA (f), Orai3 shRNA (g), TrpC1 shRNA (h), Orai1/2/3 shRNA (i), Stim1 shRNA (j). Scale bar indicates 0.5 for y axis and 100s for x axis. Tg: thapsigargin.

    Techniques Used: Imaging, Inverted Microscopy, shRNA, Transfection, Expressing, Cell Culture

    3) Product Images from "Asynchronous Ca2+ current conducted by voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels and its implications for asynchronous neurotransmitter release"

    Article Title: Asynchronous Ca2+ current conducted by voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels and its implications for asynchronous neurotransmitter release

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

    doi: 10.1073/pnas.1121103109

    I Async in the presence of channel and pump inhibitors. ( A – F ) Averaged currents elicited by a 1-s depolarization to +30 mV normalized to the peak of the current during depolarization in control (black) and the presence of a specific ion channel or ion pump inhibitor (red). ( G – L ) Mean (±SEM) values measured from currents in A – F . Black, control; red, with inhibitors. ( A and G ) 10 mM tetraethylammonium (TEA). ( B and H ) 100 nM apamin. ( C and I ) 100 μM 2-APB. ( D and J ) 100 μM ZD-7288. ( E and K ) 10 μM ryanodine. ( F and L ) 2 μM thapsigargin.
    Figure Legend Snippet: I Async in the presence of channel and pump inhibitors. ( A – F ) Averaged currents elicited by a 1-s depolarization to +30 mV normalized to the peak of the current during depolarization in control (black) and the presence of a specific ion channel or ion pump inhibitor (red). ( G – L ) Mean (±SEM) values measured from currents in A – F . Black, control; red, with inhibitors. ( A and G ) 10 mM tetraethylammonium (TEA). ( B and H ) 100 nM apamin. ( C and I ) 100 μM 2-APB. ( D and J ) 100 μM ZD-7288. ( E and K ) 10 μM ryanodine. ( F and L ) 2 μM thapsigargin.

    Techniques Used:

    4) Product Images from "Ryanodine receptors are uncoupled from contraction in rat vena cava"

    Article Title: Ryanodine receptors are uncoupled from contraction in rat vena cava

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2012.10.006

    (a–c) Representative tracings of rat aorta and vena cava contraction, after sarcoplasmic Ca 2+ stores depletion and upon exposure to Ca 2+ -replete physiological salt solution. Tissues were incubated in Ca 2+ -free buffer for 15 min before addition of thapsigargin (1 μM) to inhibit SERCA-mediated Ca 2+ reuptake. Tissues were then incubated for 1 h before reintroduction of Ca 2+ . Shown are responses from tissues incubated with vehicle (a, c) and 1 μM thapsigargin (b, d). (e) Summary bar graphs indicating the maximum contractile response after reintroduction of Ca 2+ -replete physiological salt solution in aorta (RA) and vena cava (RVC). Black bars represent vehicle-exposed tissues. White bars represent tissues exposed to thapsigargin (1 μM). N = 4; * p
    Figure Legend Snippet: (a–c) Representative tracings of rat aorta and vena cava contraction, after sarcoplasmic Ca 2+ stores depletion and upon exposure to Ca 2+ -replete physiological salt solution. Tissues were incubated in Ca 2+ -free buffer for 15 min before addition of thapsigargin (1 μM) to inhibit SERCA-mediated Ca 2+ reuptake. Tissues were then incubated for 1 h before reintroduction of Ca 2+ . Shown are responses from tissues incubated with vehicle (a, c) and 1 μM thapsigargin (b, d). (e) Summary bar graphs indicating the maximum contractile response after reintroduction of Ca 2+ -replete physiological salt solution in aorta (RA) and vena cava (RVC). Black bars represent vehicle-exposed tissues. White bars represent tissues exposed to thapsigargin (1 μM). N = 4; * p

    Techniques Used: Incubation

    5) Product Images from "Ethylatropine Bromide as a Peripherally Restricted Muscarinic Antagonist"

    Article Title: Ethylatropine Bromide as a Peripherally Restricted Muscarinic Antagonist

    Journal: ACS chemical neuroscience

    doi: 10.1021/acschemneuro.6b00334

    Ethylatropine bromide inhibits the human M1 receptor. (A) Ca 2+ -dependent fluorescent signal of CHO-hM1 cells loaded with Fluo-4 was monitored. With use of the integrated fluidics of the FLEXStation II, a bolus of HBSS was delivered at 60 s, resulting in a miniscule reduction in the fluorescent signal. Addition of ATP (100 μ M) or thapsigargin (1 μ M) at 180 s resulted in a long-lasting increase in [Ca 2+ ] i . Data are the mean of eight replicates from a single plate. Bars indicate the duration of drug exposures. (B) A rapid increase in fluorescence was detected after application of the muscarinic receptor agonist carbachol at various concentrations. Data are the mean of eight replicates from a single plate for each concentration applied. (C) Concentration–response curves of the carbachol and pilocarpine-induced Ca 2+ signal. The maximum Ca 2+ signal was measured and the response was calculated for increasing concentrations of each agonist (carbachol, EC 50 = 34 nM, n = 3 experiments in replicates of 8; pilocarpine, EC 50 = 96 nM, n = 5 experiments in replicates of 8). (D) Carbachol induces a rapid rise in intracellular Ca 2+ at 3 μ M, which can be attenuated in a concentration-dependent manner by pretreatment with atropine (IC 50 = 48 nM, n = 3 experiments in replicates of 4), methylatropine bromide (MAB) (IC 50 = 6 nM, n = 7 experiments in replicates of 4) or ethylatropine bromide (EAB) (IC 50 = 157 nM, n = 3 experiments in replicates of 4), but not by HBSS (not shown). In (C) and (D), data are mean ± SEM.
    Figure Legend Snippet: Ethylatropine bromide inhibits the human M1 receptor. (A) Ca 2+ -dependent fluorescent signal of CHO-hM1 cells loaded with Fluo-4 was monitored. With use of the integrated fluidics of the FLEXStation II, a bolus of HBSS was delivered at 60 s, resulting in a miniscule reduction in the fluorescent signal. Addition of ATP (100 μ M) or thapsigargin (1 μ M) at 180 s resulted in a long-lasting increase in [Ca 2+ ] i . Data are the mean of eight replicates from a single plate. Bars indicate the duration of drug exposures. (B) A rapid increase in fluorescence was detected after application of the muscarinic receptor agonist carbachol at various concentrations. Data are the mean of eight replicates from a single plate for each concentration applied. (C) Concentration–response curves of the carbachol and pilocarpine-induced Ca 2+ signal. The maximum Ca 2+ signal was measured and the response was calculated for increasing concentrations of each agonist (carbachol, EC 50 = 34 nM, n = 3 experiments in replicates of 8; pilocarpine, EC 50 = 96 nM, n = 5 experiments in replicates of 8). (D) Carbachol induces a rapid rise in intracellular Ca 2+ at 3 μ M, which can be attenuated in a concentration-dependent manner by pretreatment with atropine (IC 50 = 48 nM, n = 3 experiments in replicates of 4), methylatropine bromide (MAB) (IC 50 = 6 nM, n = 7 experiments in replicates of 4) or ethylatropine bromide (EAB) (IC 50 = 157 nM, n = 3 experiments in replicates of 4), but not by HBSS (not shown). In (C) and (D), data are mean ± SEM.

    Techniques Used: Fluorescence, Concentration Assay

    6) Product Images from "A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli"

    Article Title: A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008925

    Taste-evoked Ca 2+ release in BR cells from IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. A) Bitter-evoked taste responses (5mM denatonium, n = 6) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B, n = 7) as well as the PLC blocker U73122 (C, n = 7). Similar results were obtained for sweet stimuli (20mM sucralose, n = 6) in Ca 2+ -free (D), thapsigargin (E, n = 4) and U73122 (F, n = 6) as well as for umami stimuli (10mM MPG) in Ca 2+ -free (G, n = 7), thapsigargin (H, n = 4) and U73122 (I, n = 4). Representative data for each experiment are shown in S3 Fig . Comparisons of the response amplitudes for each taste stimulus found no significant differences in the control responses between experiments (One way ANOVA, Den, p = 0.933; Suc, p = 0.623; MPG, p = 0.134).
    Figure Legend Snippet: Taste-evoked Ca 2+ release in BR cells from IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. A) Bitter-evoked taste responses (5mM denatonium, n = 6) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B, n = 7) as well as the PLC blocker U73122 (C, n = 7). Similar results were obtained for sweet stimuli (20mM sucralose, n = 6) in Ca 2+ -free (D), thapsigargin (E, n = 4) and U73122 (F, n = 6) as well as for umami stimuli (10mM MPG) in Ca 2+ -free (G, n = 7), thapsigargin (H, n = 4) and U73122 (I, n = 4). Representative data for each experiment are shown in S3 Fig . Comparisons of the response amplitudes for each taste stimulus found no significant differences in the control responses between experiments (One way ANOVA, Den, p = 0.933; Suc, p = 0.623; MPG, p = 0.134).

    Techniques Used: Mouse Assay, Planar Chromatography, Activity Assay

    7) Product Images from "CHOP and caspase 3 induction underlie glioblastoma cell death in response to endoplasmic reticulum stress"

    Article Title: CHOP and caspase 3 induction underlie glioblastoma cell death in response to endoplasmic reticulum stress

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2011.422

    Dose response of thapsigargin and tunicamycin on A172 and U373 glioblastoma cells. Vehicle-treated (DMSO) control cells (white bars); 10 μM (solid black bars); 5 μM (hatched bars); 1 μM (graybars). Data shown are representative
    Figure Legend Snippet: Dose response of thapsigargin and tunicamycin on A172 and U373 glioblastoma cells. Vehicle-treated (DMSO) control cells (white bars); 10 μM (solid black bars); 5 μM (hatched bars); 1 μM (graybars). Data shown are representative

    Techniques Used:

    Clonogenic survival of U373 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet
    Figure Legend Snippet: Clonogenic survival of U373 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet

    Techniques Used: Staining

    Motility of A172 (left) and U373 (right) glioblastoma cells treated with the endoplasmic reticulum stress inducers, thapsigargin and tunicamycin. Motility was determined with boyden chamber assays and the results are shown as micrographs of glioblastoma
    Figure Legend Snippet: Motility of A172 (left) and U373 (right) glioblastoma cells treated with the endoplasmic reticulum stress inducers, thapsigargin and tunicamycin. Motility was determined with boyden chamber assays and the results are shown as micrographs of glioblastoma

    Techniques Used:

    Assessment of caspase 3 activity in A172 and U373 glioblastoma cells in response to thapsigargin (1 μM) and tunicamycin (1 μM) exposure. Caspase protein activity was measured 48 h post-exposure to thapsigargin or tunicamycin (vehicle control,
    Figure Legend Snippet: Assessment of caspase 3 activity in A172 and U373 glioblastoma cells in response to thapsigargin (1 μM) and tunicamycin (1 μM) exposure. Caspase protein activity was measured 48 h post-exposure to thapsigargin or tunicamycin (vehicle control,

    Techniques Used: Activity Assay

    Clonogenic survival of A172 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet
    Figure Legend Snippet: Clonogenic survival of A172 glioblastoma cells treated with endoplasmic reticulum stress inducers. (A) Vehicle control (DMSO); (B) thapsigargin (1 μM); (C) tunicamycin (1 μM). Cells shown are of a single colony stained with crystal violet

    Techniques Used: Staining

    Time course analysis of glioblastoma cell proliferation post-exposure to thapsigargin or tunicamycin. Cells were exposed on day 0 to solvent (DMSO), thapsigargin (1 μM) or tunicamycin (1 μM) and monitored for a period of 6 days. The proliferation
    Figure Legend Snippet: Time course analysis of glioblastoma cell proliferation post-exposure to thapsigargin or tunicamycin. Cells were exposed on day 0 to solvent (DMSO), thapsigargin (1 μM) or tunicamycin (1 μM) and monitored for a period of 6 days. The proliferation

    Techniques Used:

    Western blot analysis of CHOP protein levels in glioblastoma cells treated with thapsigargin (1 μM) or tunicamyicin (1 μM). A172 cells (lanes 1–5); U373 cells (lanes 6–10). Vehicle-treated control cells (DMSO) (lanes 1
    Figure Legend Snippet: Western blot analysis of CHOP protein levels in glioblastoma cells treated with thapsigargin (1 μM) or tunicamyicin (1 μM). A172 cells (lanes 1–5); U373 cells (lanes 6–10). Vehicle-treated control cells (DMSO) (lanes 1

    Techniques Used: Western Blot

    8) Product Images from "Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation"

    Article Title: Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-70

    Bradykinin elicits ATP release from human fibroblasts by a mechanism depending on intracellular Ca 2+ mobilization. Panels A , B and D represent cells loaded with quinacrine (30 μM, a fluorescent dye that specifically binds ATP), for 60 min at 37°C. ATP release was detected by single-cell confocal microscopy in the time-lapse mode measuring the fluorescence intensity decay 4 min after bradykinin (BK, 30 μM, A and B ) application as compared to the control situation, in which only Tyrode’s solution was applied (A) . Panel D , shows the effect of BK (30 μM) after pretreatment of the cells with the selective endoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin (2 μM, Di ), and after removal of extracellular Ca 2+ (Ca 2+ -free medium plus EGTA, 100 μM, Dii ); the effect of BK (30 μM) in the presence of the selective Panx1 inhibitor, 10 Panx (100 μM, Diii ), is also shown. Image scale bars: 30 μm. Graphs show quinacrine fluorescence decay (arbitrary units, a.u.) plotted versus time in the presence of Tyrode’s solution (B) , thapsigargin (2 μM, Di ) and Ca 2+ -free medium ( Dii) . Black arrows indicate the time of drugs application. Each point represents pooled data from an n number of cells. The vertical bars represent S.E.M.. Panel C , shows the ATP content in human subcutaneous fibroblast cultures at given time intervals (0-240 seconds) in the presence of BK (30 μM, open bars) as compared to the control condition where only Tyrode’s solution was applied (closed bars). Relative luminescence units (RLUs) were calibrated using a 4 nM ATP standard (left hand-side black vertical bar). Each bar represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p
    Figure Legend Snippet: Bradykinin elicits ATP release from human fibroblasts by a mechanism depending on intracellular Ca 2+ mobilization. Panels A , B and D represent cells loaded with quinacrine (30 μM, a fluorescent dye that specifically binds ATP), for 60 min at 37°C. ATP release was detected by single-cell confocal microscopy in the time-lapse mode measuring the fluorescence intensity decay 4 min after bradykinin (BK, 30 μM, A and B ) application as compared to the control situation, in which only Tyrode’s solution was applied (A) . Panel D , shows the effect of BK (30 μM) after pretreatment of the cells with the selective endoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin (2 μM, Di ), and after removal of extracellular Ca 2+ (Ca 2+ -free medium plus EGTA, 100 μM, Dii ); the effect of BK (30 μM) in the presence of the selective Panx1 inhibitor, 10 Panx (100 μM, Diii ), is also shown. Image scale bars: 30 μm. Graphs show quinacrine fluorescence decay (arbitrary units, a.u.) plotted versus time in the presence of Tyrode’s solution (B) , thapsigargin (2 μM, Di ) and Ca 2+ -free medium ( Dii) . Black arrows indicate the time of drugs application. Each point represents pooled data from an n number of cells. The vertical bars represent S.E.M.. Panel C , shows the ATP content in human subcutaneous fibroblast cultures at given time intervals (0-240 seconds) in the presence of BK (30 μM, open bars) as compared to the control condition where only Tyrode’s solution was applied (closed bars). Relative luminescence units (RLUs) were calibrated using a 4 nM ATP standard (left hand-side black vertical bar). Each bar represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p

    Techniques Used: Confocal Microscopy, Fluorescence

    Bradykinin stimulates the release of intracellular Ca 2+ stores and Ca 2+ influx from the extracellular space. Panel A shows immunoreactivity of cells cultured from explants of human subcutaneous tissue against fibroblast-cell markers, vimentin (red, Ai ) and type I collagen (green, Ai ), and α-smooth muscle actin (SMA-FITC, green, Aii ). Negative controls, in which cells were incubated only with secondary antibodies, Alexa Fluor 488 (green) and Alexa Fluor 568 (red), are shown for comparison purposes ( Aiii ) ; a positive control of SMA-FITC immunoreactivity in rat cardiac myofibroblasts is also shown (green, Aiv ). Cell nuclei are stained with DAPI (blue); scale bar 60 μm. Panel B illustrates intracellular Ca 2+ ([Ca 2+ ] i ) oscillations in cultured human subcutaneous fibroblasts loaded with the fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods) obtained in the absence and in the presence of bradykinin (BK, 30 μM). Changes in fluorescence were detected in the time-lapse mode with a confocal microscope. Calibration to the maximal calcium load produced by ionomycin (5 μM, 100% response) is also shown for comparison. Image scale bars: 30 μm. Panel C shows that the kinetics of BK-induced [Ca 2+ ] i signals differed slightly between cells of a given population. Panel D depicts the concentration-response curve of [Ca 2+ ] i oscillations produced by BK (0.003-100 μM). Panels E , F and G , represent [Ca 2+ ] i oscillations produced by BK (30 μM) applied in the absence (E) and in the presence of the selective endoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin (2 μM, F ), and after removal of extracellular Ca 2+ (Ca 2+ -free medium plus EGTA, 100 μM, G ). Black arrows indicate the time of drugs application. Each point represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p
    Figure Legend Snippet: Bradykinin stimulates the release of intracellular Ca 2+ stores and Ca 2+ influx from the extracellular space. Panel A shows immunoreactivity of cells cultured from explants of human subcutaneous tissue against fibroblast-cell markers, vimentin (red, Ai ) and type I collagen (green, Ai ), and α-smooth muscle actin (SMA-FITC, green, Aii ). Negative controls, in which cells were incubated only with secondary antibodies, Alexa Fluor 488 (green) and Alexa Fluor 568 (red), are shown for comparison purposes ( Aiii ) ; a positive control of SMA-FITC immunoreactivity in rat cardiac myofibroblasts is also shown (green, Aiv ). Cell nuclei are stained with DAPI (blue); scale bar 60 μm. Panel B illustrates intracellular Ca 2+ ([Ca 2+ ] i ) oscillations in cultured human subcutaneous fibroblasts loaded with the fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods) obtained in the absence and in the presence of bradykinin (BK, 30 μM). Changes in fluorescence were detected in the time-lapse mode with a confocal microscope. Calibration to the maximal calcium load produced by ionomycin (5 μM, 100% response) is also shown for comparison. Image scale bars: 30 μm. Panel C shows that the kinetics of BK-induced [Ca 2+ ] i signals differed slightly between cells of a given population. Panel D depicts the concentration-response curve of [Ca 2+ ] i oscillations produced by BK (0.003-100 μM). Panels E , F and G , represent [Ca 2+ ] i oscillations produced by BK (30 μM) applied in the absence (E) and in the presence of the selective endoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin (2 μM, F ), and after removal of extracellular Ca 2+ (Ca 2+ -free medium plus EGTA, 100 μM, G ). Black arrows indicate the time of drugs application. Each point represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p

    Techniques Used: Cell Culture, Incubation, Positive Control, Staining, Fluorescence, Microscopy, Produced, Concentration Assay

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

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

    Journal: International Journal of Peptides

    doi: 10.1155/2013/573716

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

    Techniques Used: Concentration Assay

    10) Product Images from "Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines"

    Article Title: Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085367

    Effects of various inhibitors of Ca 2+ entry on DA-actin distribution. Neurons (21 DIV) were incubated in normal medium containing 50 µM APV (A), 20 mM EGTA (B), 20 µM nifedipine (C), or 1 µM thapsigargin (D) for 30 min. The neurons were then stimulated with 100 µM glutamate for an additional 10 min. F-actin images indicate that spines kept their structure during the experiment although their shapes were changed. Scale bars, 5 µm. ( A ) APV pretreatment significantly increased both the drebrin and actin SDRs (n = 30 cells; p
    Figure Legend Snippet: Effects of various inhibitors of Ca 2+ entry on DA-actin distribution. Neurons (21 DIV) were incubated in normal medium containing 50 µM APV (A), 20 mM EGTA (B), 20 µM nifedipine (C), or 1 µM thapsigargin (D) for 30 min. The neurons were then stimulated with 100 µM glutamate for an additional 10 min. F-actin images indicate that spines kept their structure during the experiment although their shapes were changed. Scale bars, 5 µm. ( A ) APV pretreatment significantly increased both the drebrin and actin SDRs (n = 30 cells; p

    Techniques Used: Incubation

    11) Product Images from "Gray Matter NG2 Cells Display Multiple Ca2+-Signaling Pathways and Highly Motile Processes"

    Article Title: Gray Matter NG2 Cells Display Multiple Ca2+-Signaling Pathways and Highly Motile Processes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017575

    CICR in NG2 cells. (A) Train stimulation (gray box, bottom trace) evoked [Ca 2+ ] i elevations in the presence of Ca 2+ -containing bath solution but not in Ca 2+ -free bath solution (0 mM Ca 2+ , 2 mM EDTA). Traces represent the average of 5 cells. (B) Single pulses (gray box, lower trace) induced [Ca 2+ ] i elevations that were sensitive to thapsigargin (1 µM, elevation decreased to 10%) indicating a contribution of Ca 2+ -release from intracellular stores. Traces represent the average of 5 cells.
    Figure Legend Snippet: CICR in NG2 cells. (A) Train stimulation (gray box, bottom trace) evoked [Ca 2+ ] i elevations in the presence of Ca 2+ -containing bath solution but not in Ca 2+ -free bath solution (0 mM Ca 2+ , 2 mM EDTA). Traces represent the average of 5 cells. (B) Single pulses (gray box, lower trace) induced [Ca 2+ ] i elevations that were sensitive to thapsigargin (1 µM, elevation decreased to 10%) indicating a contribution of Ca 2+ -release from intracellular stores. Traces represent the average of 5 cells.

    Techniques Used:

    12) Product Images from "Transactivation of epidermal growth factor receptor through platelet-activating factor/receptor in ovarian cancer cells"

    Article Title: Transactivation of epidermal growth factor receptor through platelet-activating factor/receptor in ovarian cancer cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-014-0085-6

    Role of Ca 2+ and PKC in responses to PAF in SKOV-3 cells. (A) SKOV-3 cells were pretreated for 1 h with the PLCβ inhibitor U73122 (20 μM) before stimulation with PAF (100 nM) or EGF (5 ng/ml) for 5 min. (B) SKOV-3 cells were pretreated for 1 h with the Ca 2+ chelator BAPTA-AM (20 μM) before stimulation with PAF (100 nM) or thapsigargin (1 μM) for 5 min. (C) SKOV-3 cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 nM PAF. (D) SKOV-3 cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 nM PAF in the presence or absence of WEB2086, calcium mobilization was assayed by monitoring the change in Fura-2/AM fluorescence. (E and F) SKOV-3 cells were pretreated for 1 h with the PKC inhibitor GF109203X (5 μM) before stimulation with PAF (100 nM) or PMA (1 μM) for 5 min. Cells were then harvested and subjected to Western blot analysis. The data shown are representative of at least three independent experiments. Data were analyzed by Student’s t -test. * p
    Figure Legend Snippet: Role of Ca 2+ and PKC in responses to PAF in SKOV-3 cells. (A) SKOV-3 cells were pretreated for 1 h with the PLCβ inhibitor U73122 (20 μM) before stimulation with PAF (100 nM) or EGF (5 ng/ml) for 5 min. (B) SKOV-3 cells were pretreated for 1 h with the Ca 2+ chelator BAPTA-AM (20 μM) before stimulation with PAF (100 nM) or thapsigargin (1 μM) for 5 min. (C) SKOV-3 cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 nM PAF. (D) SKOV-3 cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 nM PAF in the presence or absence of WEB2086, calcium mobilization was assayed by monitoring the change in Fura-2/AM fluorescence. (E and F) SKOV-3 cells were pretreated for 1 h with the PKC inhibitor GF109203X (5 μM) before stimulation with PAF (100 nM) or PMA (1 μM) for 5 min. Cells were then harvested and subjected to Western blot analysis. The data shown are representative of at least three independent experiments. Data were analyzed by Student’s t -test. * p

    Techniques Used: Fluorescence, Western Blot

    13) Product Images from "Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ In Vitro and In Cellulo"

    Article Title: Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ In Vitro and In Cellulo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169804

    Impact of the p70S6K1 inhibitors on LTB 4 biosynthesis and ω-oxidation in neutrophil suspensions. A-D) Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl 2 ) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes. A,C,D ) Samples were processed and analyzed for LTB 4 biosynthesis as described in methods. Data are the mean (± S.D) of 5 independent experiments, each performed in duplicate. A ) Leukotrienes represent the sum of LTB 4 , 20-OH-LTB 4 and 20-COOH-LTB 4 . C,D ) ω-LTB 4 represents the sum of 20-OH-LTB 4 and 20-COOH-LTB 4 . B ) Samples were processed and analyzed for S6 and phospho-S6 content as described in methods. Data are from one experiment, representative of three.
    Figure Legend Snippet: Impact of the p70S6K1 inhibitors on LTB 4 biosynthesis and ω-oxidation in neutrophil suspensions. A-D) Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl 2 ) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes. A,C,D ) Samples were processed and analyzed for LTB 4 biosynthesis as described in methods. Data are the mean (± S.D) of 5 independent experiments, each performed in duplicate. A ) Leukotrienes represent the sum of LTB 4 , 20-OH-LTB 4 and 20-COOH-LTB 4 . C,D ) ω-LTB 4 represents the sum of 20-OH-LTB 4 and 20-COOH-LTB 4 . B ) Samples were processed and analyzed for S6 and phospho-S6 content as described in methods. Data are from one experiment, representative of three.

    Techniques Used: Incubation

    Removal of the inhibitory constraint exerted by CYP4F3A inhibitors on LTB 4 ω-oxidation in neutrophils. Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl 2 ) were incubated with A,B) 30 μM PF-4708671 or vehicle for 5 minutes, C) 30 μM PF-4708671 for 15 minutes, D,E) 30 μM 17-ODYA for 30 minutes, or F) 30 μM 17-ODYA for 15 minutes. Neutrophils were washed (or not) with autologous plasma or HBSS-CaCl 2 as described in methods. A,D) 100 nM thapsigargin or B,C,E,F) 1 μM LTB 4 were then added for 10 and 20 minutes, respectively. Samples then were processed and analyzed for 20-OH-LTB 4 and 20-COOH-LTB 4 as described in methods. Data are the mean (± S.D) of 4 independent experiments, each performed in duplicate.
    Figure Legend Snippet: Removal of the inhibitory constraint exerted by CYP4F3A inhibitors on LTB 4 ω-oxidation in neutrophils. Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl 2 ) were incubated with A,B) 30 μM PF-4708671 or vehicle for 5 minutes, C) 30 μM PF-4708671 for 15 minutes, D,E) 30 μM 17-ODYA for 30 minutes, or F) 30 μM 17-ODYA for 15 minutes. Neutrophils were washed (or not) with autologous plasma or HBSS-CaCl 2 as described in methods. A,D) 100 nM thapsigargin or B,C,E,F) 1 μM LTB 4 were then added for 10 and 20 minutes, respectively. Samples then were processed and analyzed for 20-OH-LTB 4 and 20-COOH-LTB 4 as described in methods. Data are the mean (± S.D) of 4 independent experiments, each performed in duplicate.

    Techniques Used: Incubation

    14) Product Images from "Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light"

    Article Title: Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00561

    Light-induced store-operated Ca 2+ entry (SOCE). (A) The SOCE was observed by the [Ca 2+ ] o add-back from 0 to 2.5 mM after depletion of the intracellular Ca 2+ store by thapsigargin (TG, orange stripe, 5 μM), while the change of R-GECO1 fluorescence (Δ F / F 0 ) was recorded (0.1 fps) from a ChRGR ER -expressing C2C12 cell. (B) Similar to (A) , but the OS (blue stripe, 475 ± 10 nm, 10 ms pulse at 20 Hz for 0.5 s) was repetitively applied (50 cycles at every 10 s). Note that the OS increased the fluorescence only slightly because it was applied between the sampling of images. (C) Summary of the OS-induced SOCE: without add-back [Ca 2+ ] o after OS (left, n = 7), with add-back [Ca 2+ ] o after OS ( n = 12) and with add-back [Ca 2+ ] o after TG ( n = 8). ‡ P
    Figure Legend Snippet: Light-induced store-operated Ca 2+ entry (SOCE). (A) The SOCE was observed by the [Ca 2+ ] o add-back from 0 to 2.5 mM after depletion of the intracellular Ca 2+ store by thapsigargin (TG, orange stripe, 5 μM), while the change of R-GECO1 fluorescence (Δ F / F 0 ) was recorded (0.1 fps) from a ChRGR ER -expressing C2C12 cell. (B) Similar to (A) , but the OS (blue stripe, 475 ± 10 nm, 10 ms pulse at 20 Hz for 0.5 s) was repetitively applied (50 cycles at every 10 s). Note that the OS increased the fluorescence only slightly because it was applied between the sampling of images. (C) Summary of the OS-induced SOCE: without add-back [Ca 2+ ] o after OS (left, n = 7), with add-back [Ca 2+ ] o after OS ( n = 12) and with add-back [Ca 2+ ] o after TG ( n = 8). ‡ P

    Techniques Used: Fluorescence, Expressing, Mass Spectrometry, Sampling

    15) Product Images from "Presynaptic residual calcium and synaptic facilitation at hippocampal synapses of mice with altered expression of SNAP-25"

    Article Title: Presynaptic residual calcium and synaptic facilitation at hippocampal synapses of mice with altered expression of SNAP-25

    Journal: Brain research

    doi: 10.1016/j.brainres.2011.10.035

    A. Percent change of ∫ΔF/F 0 measured with MgGreen following a single pulse in 3 μM thapsigargin compared to the ∫ΔF/F 0 response in ACSF in the same slice (n = 4). (Thapsigargin was dissolved in DMSO and an equivalent
    Figure Legend Snippet: A. Percent change of ∫ΔF/F 0 measured with MgGreen following a single pulse in 3 μM thapsigargin compared to the ∫ΔF/F 0 response in ACSF in the same slice (n = 4). (Thapsigargin was dissolved in DMSO and an equivalent

    Techniques Used:

    16) Product Images from "FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk"

    Article Title: FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2016.01.001

    Ca 2+ /PKA crosstalk does not occur in the presence of a Ca 2+ -resistant gravin mutant. Cartoons depict the AKAP18α-gravin chimera, which replaces gravin’s membrane localization domains with the first twelve amino acids of AKAP18α. Images and corresponding linescans of AN3 CA cells before and after thapsigargin treatment show that while WT gravin undergoes redistribution, the AKAP18-gravin chimera does not. Graph C shows the quantification of the fold change in membrane:cytosolic fluoresence intensity before and after thapsigargin treatment as depicted in the corresponding linescans. Note that a value of 1.0 depicts no redistribution after thapsigargin treatment, and values higher than 1.0 depict a greater extent of redistribution. Graphs D–E show that both WT gravin and AKAP18-gravin caused elevated PKA activity levels at the plasma membrane compared with controls lacking gravin. Graphs F–G show that thapsigargin had no effect on PKA activity levels at the plasma membrane in cells expressing AKAP18-gravin. Statistical differences were determined by a T-test in C, G or one-way ANOVA with Holm-Sidak post hoc in E and are indicated by asterisks. Error bars denote SD. Scale bar = 20 μm.
    Figure Legend Snippet: Ca 2+ /PKA crosstalk does not occur in the presence of a Ca 2+ -resistant gravin mutant. Cartoons depict the AKAP18α-gravin chimera, which replaces gravin’s membrane localization domains with the first twelve amino acids of AKAP18α. Images and corresponding linescans of AN3 CA cells before and after thapsigargin treatment show that while WT gravin undergoes redistribution, the AKAP18-gravin chimera does not. Graph C shows the quantification of the fold change in membrane:cytosolic fluoresence intensity before and after thapsigargin treatment as depicted in the corresponding linescans. Note that a value of 1.0 depicts no redistribution after thapsigargin treatment, and values higher than 1.0 depict a greater extent of redistribution. Graphs D–E show that both WT gravin and AKAP18-gravin caused elevated PKA activity levels at the plasma membrane compared with controls lacking gravin. Graphs F–G show that thapsigargin had no effect on PKA activity levels at the plasma membrane in cells expressing AKAP18-gravin. Statistical differences were determined by a T-test in C, G or one-way ANOVA with Holm-Sidak post hoc in E and are indicated by asterisks. Error bars denote SD. Scale bar = 20 μm.

    Techniques Used: Mutagenesis, Activity Assay, Expressing

    Receptor-mediated Ca 2+ elevation modulates PKA activity by gravin redistribution, and gravin regulates β-adrenergic receptor mediated PKA activity at the plasma membrane. Fluorescent images show that ATP caused the redistribution of gravin away from the plasma membrane. Graphs A,B show that gravin raised forskolin-stimulated PKA activity at the membrane, but this increase was not observed in cells expressing gravin and pretreated with ATP (10mM) for 30 min. ATP pretreatment had no effect on forskolin-stimulated PKA activity in the absence of gravin. Graphs C,D show that gravin increased isoproterenol-stimulated PKA activity levels at the plasma membrane compared with control cells lacking gravin. This increase was not observed in cells expressing gravin but pretreated with thapsigargin (10 μm for 30 min). Thapsigargin pretreatment had no effect on isoproterenol-stimulated PKA activity in the absence of gravin. One-way ANOVA with Kruskal-Wallis post hoc showed significant differences between the treatments as indicated by asterisks. Error bars denote SD. Scale bar = 10 μm.
    Figure Legend Snippet: Receptor-mediated Ca 2+ elevation modulates PKA activity by gravin redistribution, and gravin regulates β-adrenergic receptor mediated PKA activity at the plasma membrane. Fluorescent images show that ATP caused the redistribution of gravin away from the plasma membrane. Graphs A,B show that gravin raised forskolin-stimulated PKA activity at the membrane, but this increase was not observed in cells expressing gravin and pretreated with ATP (10mM) for 30 min. ATP pretreatment had no effect on forskolin-stimulated PKA activity in the absence of gravin. Graphs C,D show that gravin increased isoproterenol-stimulated PKA activity levels at the plasma membrane compared with control cells lacking gravin. This increase was not observed in cells expressing gravin but pretreated with thapsigargin (10 μm for 30 min). Thapsigargin pretreatment had no effect on isoproterenol-stimulated PKA activity in the absence of gravin. One-way ANOVA with Kruskal-Wallis post hoc showed significant differences between the treatments as indicated by asterisks. Error bars denote SD. Scale bar = 10 μm.

    Techniques Used: Activity Assay, Expressing

    Thapsigargin triggers gravin redistribution and abolishes gravin-mediated elevation in plasma membrane PKA activity. Representative fluorescent micrographs (A) illustrate the redistribution of gravin-EGFP away from the plasma membrane by thapsigargin. Gravin-V5/His redistribution was confirmed after FRET experiments by quantifying the percentage of cells showing gravin localization at the membrane, shown in the box plot represented in B (Mann-Whitney Rank Sum Test, p
    Figure Legend Snippet: Thapsigargin triggers gravin redistribution and abolishes gravin-mediated elevation in plasma membrane PKA activity. Representative fluorescent micrographs (A) illustrate the redistribution of gravin-EGFP away from the plasma membrane by thapsigargin. Gravin-V5/His redistribution was confirmed after FRET experiments by quantifying the percentage of cells showing gravin localization at the membrane, shown in the box plot represented in B (Mann-Whitney Rank Sum Test, p

    Techniques Used: Activity Assay, MANN-WHITNEY

    17) Product Images from "Functional expression of the Ca2+ signaling machinery in human embryonic stem cells"

    Article Title: Functional expression of the Ca2+ signaling machinery in human embryonic stem cells

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2017.29

    Ca 2+ entry machinery of the plasma membrane and the protein expression of VOCCs in H9 and H7 hESCs. (A) [Ca 2+ ] i recording in hESCs with a high K + concentration (145 mmol/L) in the extracellular solution. (B) Western blot analysis of T-type Ca 2+ channels Cav3.1 and Cav3.2 in H7 and H9 cells. 293FT was used as a positive control. Data shown are representative of three independent experiments. (C) Western blot analysis of L-type Ca 2+ channels Cav1.2, Cav1.3 and Cav1.4 in H7 and H9 cells. 293FT was used as a positive control. Data shown are representative of three independent experiments. α-Tub, α-tubulin, an internal control. (D) An outline of the experimental protocol for measuring SOCE. (E) Representative traces of Ca 2+ signals in hESCs with the various treatments as shown in (D). TG, thapsigargin (1 μmol/L); ATP, 100 μmol/L. Consistent data were obtained from three independent experiments and 50 hESCs in each experiment from each cell line.
    Figure Legend Snippet: Ca 2+ entry machinery of the plasma membrane and the protein expression of VOCCs in H9 and H7 hESCs. (A) [Ca 2+ ] i recording in hESCs with a high K + concentration (145 mmol/L) in the extracellular solution. (B) Western blot analysis of T-type Ca 2+ channels Cav3.1 and Cav3.2 in H7 and H9 cells. 293FT was used as a positive control. Data shown are representative of three independent experiments. (C) Western blot analysis of L-type Ca 2+ channels Cav1.2, Cav1.3 and Cav1.4 in H7 and H9 cells. 293FT was used as a positive control. Data shown are representative of three independent experiments. α-Tub, α-tubulin, an internal control. (D) An outline of the experimental protocol for measuring SOCE. (E) Representative traces of Ca 2+ signals in hESCs with the various treatments as shown in (D). TG, thapsigargin (1 μmol/L); ATP, 100 μmol/L. Consistent data were obtained from three independent experiments and 50 hESCs in each experiment from each cell line.

    Techniques Used: Expressing, Concentration Assay, Western Blot, Positive Control

    18) Product Images from "Intracellular Calcium Regulation by Burst Discharge Determines Bidirectional Long-Term Synaptic Plasticity at the Cerebellum Input Stage"

    Article Title: Intracellular Calcium Regulation by Burst Discharge Determines Bidirectional Long-Term Synaptic Plasticity at the Cerebellum Input Stage

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0410-05.2005

    Effect of thapsigargin on [Ca 2+ ] i transients. [Ca 2+ ] i transients were induced by a 200 ms depolarization from -70 to 0 mV after 30 min of slice preincubation with 500 n m thapsigargin. A , The depolarization-induced [Ca 2+ ] i transient (continuous line) shows
    Figure Legend Snippet: Effect of thapsigargin on [Ca 2+ ] i transients. [Ca 2+ ] i transients were induced by a 200 ms depolarization from -70 to 0 mV after 30 min of slice preincubation with 500 n m thapsigargin. A , The depolarization-induced [Ca 2+ ] i transient (continuous line) shows

    Techniques Used: Mass Spectrometry

    19) Product Images from "The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine"

    Article Title: The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2018.01.003

    Effect of FCCP and antimycin on I CRAC . I CRAC was measured in HEK 293 cells expressing Orai1 and Stim1 using whole-cell voltage-clamp (holding potential = −50 mV). Cells were treated with Ca 2+ -free PSS containing thapsigargin (1 μM) for 10 min to deplete ER Ca 2+ . A shows the response elicited by reintroduction of 2 mM [Ca 2+ ] o : an inward current was activated and FCCP (1 μM) had no effect on this current. Ramp potentials from −80 to +60 mV were run during the experiment (examples noted by a , b and c in th trace in A ) B shows responses to ramp potentials in Panel A ( a is control, black trace, b is after addition of 2 mM [Ca 2+ ] o , red trace, and c is after addition of FCCP (1 μM), green trace). C shows Antimycin had no effect on the current activated by reintroduction of 2 mM [Ca 2+ ] o . D shows responses to ramp potentials in Panel C ( a is control, black trace, b is after addition of 2 mM [Ca 2+ ] o , red trace, and c is after addition of Antimycin A (10 μM), green trace). E Summary data showing the effects of FCCP and Antimycin A on I CRAC at −80 mV (n=6, ***p
    Figure Legend Snippet: Effect of FCCP and antimycin on I CRAC . I CRAC was measured in HEK 293 cells expressing Orai1 and Stim1 using whole-cell voltage-clamp (holding potential = −50 mV). Cells were treated with Ca 2+ -free PSS containing thapsigargin (1 μM) for 10 min to deplete ER Ca 2+ . A shows the response elicited by reintroduction of 2 mM [Ca 2+ ] o : an inward current was activated and FCCP (1 μM) had no effect on this current. Ramp potentials from −80 to +60 mV were run during the experiment (examples noted by a , b and c in th trace in A ) B shows responses to ramp potentials in Panel A ( a is control, black trace, b is after addition of 2 mM [Ca 2+ ] o , red trace, and c is after addition of FCCP (1 μM), green trace). C shows Antimycin had no effect on the current activated by reintroduction of 2 mM [Ca 2+ ] o . D shows responses to ramp potentials in Panel C ( a is control, black trace, b is after addition of 2 mM [Ca 2+ ] o , red trace, and c is after addition of Antimycin A (10 μM), green trace). E Summary data showing the effects of FCCP and Antimycin A on I CRAC at −80 mV (n=6, ***p

    Techniques Used: Expressing

    20) Product Images from "Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion"

    Article Title: Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059179

    Involvement of Ca 2+ store. A , Typical [ ΔF/F ] B response of a calyx to a single 20 ms laser pulse in the cation-free extracellular solution (black), the response with additional xestospongin C (blue), the response with additional dantrolene (red) and the response after repetitive photostimulation with additional thapsigargin (green). Each trace is an average of five consecutive records. B , Summary of peak [ ΔF/F ] B changes (mean ± SEM) in the cation-free solution. Each column indicates the relative value to that without any pharmacological reagents. *, P
    Figure Legend Snippet: Involvement of Ca 2+ store. A , Typical [ ΔF/F ] B response of a calyx to a single 20 ms laser pulse in the cation-free extracellular solution (black), the response with additional xestospongin C (blue), the response with additional dantrolene (red) and the response after repetitive photostimulation with additional thapsigargin (green). Each trace is an average of five consecutive records. B , Summary of peak [ ΔF/F ] B changes (mean ± SEM) in the cation-free solution. Each column indicates the relative value to that without any pharmacological reagents. *, P

    Techniques Used: Mass Spectrometry

    21) Product Images from "UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells"

    Article Title: UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00429

    Post-receptor signaling pathways linked to UTP-Ca 2+ responses in BON cells. Representative Ca 2+ responses showing that (A) 100 μM La 3+ , (B) 10 μM 2APB or (C) 1 μM thapsigargin (thaps) can block UTP-induced Ca 2+ transients. In Contrast, (D) RuRed, (E) 20 μM MRS1845 or (F) 10 μM nicardipine are not effective in blocking UTP-induced Ca 2+ transients. After washing-out the drug (15 min) a partial recovery of the UTP-response was seen for La 3+ and 2APB but not for thapsigargin (thaps). (G–L) Pooled data for effects of each of the treatments illustrated in (A–F) indicating that La 3+ , 2APB or thaps can abolish Ca 2+ transients ( p
    Figure Legend Snippet: Post-receptor signaling pathways linked to UTP-Ca 2+ responses in BON cells. Representative Ca 2+ responses showing that (A) 100 μM La 3+ , (B) 10 μM 2APB or (C) 1 μM thapsigargin (thaps) can block UTP-induced Ca 2+ transients. In Contrast, (D) RuRed, (E) 20 μM MRS1845 or (F) 10 μM nicardipine are not effective in blocking UTP-induced Ca 2+ transients. After washing-out the drug (15 min) a partial recovery of the UTP-response was seen for La 3+ and 2APB but not for thapsigargin (thaps). (G–L) Pooled data for effects of each of the treatments illustrated in (A–F) indicating that La 3+ , 2APB or thaps can abolish Ca 2+ transients ( p

    Techniques Used: Blocking Assay

    UTP evokes a K v 7 and PLC-dependent V m depolarization. (A,B,D) UTP depolarization was eliminated in the presence of a PLC inhibitor U73122 but not with the inactive compound U73343. (C,E) Blockade of K v 7.1/7.2/7.3 potassium channels by XE-991 depolarizes the V m and increases R i in a similar manner to UTP (5.13 ± 0.64 mV for UTP vs. 6.08 ± 0.82 mV for XE-991). (C,F) Pre-incubation with 10 μM XE-991 reduced the number of cells responsive to UTP ( p = 0.02). (G) Preincubation with La 3+ inhibited the effect of UTP on membrane potential ( ∗ p = 0.03). UTP-evoked V m depolarization was not affected by treating cells with thapsigargin, GF109203X, and Ca 2+ free external solution.
    Figure Legend Snippet: UTP evokes a K v 7 and PLC-dependent V m depolarization. (A,B,D) UTP depolarization was eliminated in the presence of a PLC inhibitor U73122 but not with the inactive compound U73343. (C,E) Blockade of K v 7.1/7.2/7.3 potassium channels by XE-991 depolarizes the V m and increases R i in a similar manner to UTP (5.13 ± 0.64 mV for UTP vs. 6.08 ± 0.82 mV for XE-991). (C,F) Pre-incubation with 10 μM XE-991 reduced the number of cells responsive to UTP ( p = 0.02). (G) Preincubation with La 3+ inhibited the effect of UTP on membrane potential ( ∗ p = 0.03). UTP-evoked V m depolarization was not affected by treating cells with thapsigargin, GF109203X, and Ca 2+ free external solution.

    Techniques Used: Planar Chromatography, Incubation

    22) Product Images from "Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA"

    Article Title: Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00195

    Facilitation of glutamate release mediated by presynaptic kainate receptor (KAR) activation requires an increase of Ca 2+ in the cytosol at PF-PuC synapses. (A) Time-course of KA (3 μM) effect on eEPSCs amplitude in control condition (circles) and in slices treated with philanthotoxin (squares). (B) Quantification of modulation observed in (A) . (C) Time-course of the effect of KA on eEPSCs amplitude in control slices (circles) and in thapsigargin-treated slices (squares). (D) In slices treated with thapsigargin or ryanodine, the increase of eEPSCs amplitude induced by KA is prevented. The number of slices (from two to three mice) is indicated in parenthesis at the top of each bar. Results are expressed as means ± SEM (** P
    Figure Legend Snippet: Facilitation of glutamate release mediated by presynaptic kainate receptor (KAR) activation requires an increase of Ca 2+ in the cytosol at PF-PuC synapses. (A) Time-course of KA (3 μM) effect on eEPSCs amplitude in control condition (circles) and in slices treated with philanthotoxin (squares). (B) Quantification of modulation observed in (A) . (C) Time-course of the effect of KA on eEPSCs amplitude in control slices (circles) and in thapsigargin-treated slices (squares). (D) In slices treated with thapsigargin or ryanodine, the increase of eEPSCs amplitude induced by KA is prevented. The number of slices (from two to three mice) is indicated in parenthesis at the top of each bar. Results are expressed as means ± SEM (** P

    Techniques Used: Activation Assay, Mouse Assay

    23) Product Images from "Tonic inhibition of murine proximal colon is due to nitrergic suppression of Ca2+ signaling in interstitial cells of Cajal"

    Article Title: Tonic inhibition of murine proximal colon is due to nitrergic suppression of Ca2+ signaling in interstitial cells of Cajal

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39729-7

    Effects of thapsigargin on contractions after TTX, L-NNA or ODQ. ( A ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after TTX (1 μM). ( B ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after L-NNA (100 μM). ( C ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after ODQ (10 μM). ( D ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after TTX (1 μM; n = 15). ( E ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after L-NNA (100 μM; n = 14). ( F ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after ODQ (10 μM; n = 13). #### P
    Figure Legend Snippet: Effects of thapsigargin on contractions after TTX, L-NNA or ODQ. ( A ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after TTX (1 μM). ( B ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after L-NNA (100 μM). ( C ) Representative trace showing the effects of thapsigargin (1–10 μM) on the contractions of proximal colon after ODQ (10 μM). ( D ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after TTX (1 μM; n = 15). ( E ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after L-NNA (100 μM; n = 14). ( F ) Summarized data showing the effects of thapsigargin (1–10 μM) on contractions after ODQ (10 μM; n = 13). #### P

    Techniques Used:

    24) Product Images from "Differential Regulation of GnRH Secretion in the Preoptic Area (POA) and the Median Eminence (ME) in Male Mice"

    Article Title: Differential Regulation of GnRH Secretion in the Preoptic Area (POA) and the Median Eminence (ME) in Male Mice

    Journal: Endocrinology

    doi: 10.1210/en.2014-1458

    Increasing intracellular Ca 2+ by blocking SERCA pumps elicits GnRH release in the POA and ME but requires action potentials only in the ME. A, Experimental timeline for B and C. B and C, Representative examples of thapsigargin (Tp)-evoked GnRH release shown as a current heat map during the control period (left) and TTX treatment (right) in the POA (B) and ME (C). D, top, Experimental timeline. Bottom, Repeated injection of Tp under control conditions induces repeated GnRH release in the ME. E, Mean ± SEM peak GnRH release amplitude (left) shown as change in concentration and release duration (right). Numbers within bars indicate sample size; ' indicates second injection; “ indicates third injection of CPA, accordingly. F, Mean ± SEM percent responding to Tp (left), concentration change (center), and release duration (right). Gray bars show POA, black bars show ME; lower case letters indicate P
    Figure Legend Snippet: Increasing intracellular Ca 2+ by blocking SERCA pumps elicits GnRH release in the POA and ME but requires action potentials only in the ME. A, Experimental timeline for B and C. B and C, Representative examples of thapsigargin (Tp)-evoked GnRH release shown as a current heat map during the control period (left) and TTX treatment (right) in the POA (B) and ME (C). D, top, Experimental timeline. Bottom, Repeated injection of Tp under control conditions induces repeated GnRH release in the ME. E, Mean ± SEM peak GnRH release amplitude (left) shown as change in concentration and release duration (right). Numbers within bars indicate sample size; ' indicates second injection; “ indicates third injection of CPA, accordingly. F, Mean ± SEM percent responding to Tp (left), concentration change (center), and release duration (right). Gray bars show POA, black bars show ME; lower case letters indicate P

    Techniques Used: Blocking Assay, Injection, Concentration Assay

    25) Product Images from "Inhibition of the Ca2+ release channel, IP3R subtype 3 by caffeine slows glioblastoma invasion and migration and extends survival"

    Article Title: Inhibition of the Ca2+ release channel, IP3R subtype 3 by caffeine slows glioblastoma invasion and migration and extends survival

    Journal: Cancer Research

    doi: 10.1158/0008-5472.CAN-09-2886

    Caffeine slows motility, invasion, and colony formation of glioblastoma cells A. Monolayers of glioblastoma cells were wounded by a scrape (black box) and treated with 10 mM caffeine (Caf), 1 μM thapsigargin (Thap), or 10 μM ryanodine (Rya). All error bars represent SEM. (*p
    Figure Legend Snippet: Caffeine slows motility, invasion, and colony formation of glioblastoma cells A. Monolayers of glioblastoma cells were wounded by a scrape (black box) and treated with 10 mM caffeine (Caf), 1 μM thapsigargin (Thap), or 10 μM ryanodine (Rya). All error bars represent SEM. (*p

    Techniques Used:

    26) Product Images from "IGF-I Governs Cortical Inhibitory Synaptic Plasticity By Astrocyte Activation"

    Article Title: IGF-I Governs Cortical Inhibitory Synaptic Plasticity By Astrocyte Activation

    Journal: bioRxiv

    doi: 10.1101/2020.02.11.942532

    iLTD IGFI requires A 2A receptor activation A. Left. Same as 1B left in ACSF but in the presence of SCH or CPT (purple and blue circles respectively). Note that the A 2A receptor antagonist, SCH, prevents the iLTD IGFI . Right. Representative IPSCs in SCH and CPT before and during IGF-I. B. Same as 1D left but in the presence of SCH and CPT. C. Left. Bar plot of the calcium oscillation frequency in astrocytes in ACSF, SCH or CPT, before and during IGF-I. Right. Representative calcium traces in astrocytes under SCH or CPT, before and during IGF-I. D. Bar plot of ATP concentration in culture of astrocytes before (control) and during IGF-I exposure, and in IGF-I + NVP, IGF-I + BAPTA-AM and IGF-I + thapsigargin. E. Bar plot of ATP concentration culture of astrocytes before (control) and during IGF-I exposure from IGF-IR WT (yellow bar), IGF-IR -/- mice (green bar) and IP 3 R2 -/- mice (gold bar) .
    Figure Legend Snippet: iLTD IGFI requires A 2A receptor activation A. Left. Same as 1B left in ACSF but in the presence of SCH or CPT (purple and blue circles respectively). Note that the A 2A receptor antagonist, SCH, prevents the iLTD IGFI . Right. Representative IPSCs in SCH and CPT before and during IGF-I. B. Same as 1D left but in the presence of SCH and CPT. C. Left. Bar plot of the calcium oscillation frequency in astrocytes in ACSF, SCH or CPT, before and during IGF-I. Right. Representative calcium traces in astrocytes under SCH or CPT, before and during IGF-I. D. Bar plot of ATP concentration in culture of astrocytes before (control) and during IGF-I exposure, and in IGF-I + NVP, IGF-I + BAPTA-AM and IGF-I + thapsigargin. E. Bar plot of ATP concentration culture of astrocytes before (control) and during IGF-I exposure from IGF-IR WT (yellow bar), IGF-IR -/- mice (green bar) and IP 3 R2 -/- mice (gold bar) .

    Techniques Used: Activation Assay, Concentration Assay, Mouse Assay

    27) Product Images from "Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion"

    Article Title: Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201409071

    CaM was involved in P2X4-mediated vacuolation. (A) A CaM inhibitor, 10 µM W7 (1 h) significantly suppressed MA-induced vacuolation in Cos1 cells expressing rP2X4-GFP. Summary of vacuolated cells is shown on the right. Vacuolation was induced by either MA or Baf A1 as indicated. (B) Vacuolation of rP2X4-GFP expressing Cos1 cells was enhanced by coexpression of WT CaM but suppressed by that of CaM(1–4), a dominant-negative CaM mutant. Bar graph at right shows percentage of vacuolated cells under conditions indicated. (C) Coexpression of CaM(1–4) dramatically reduced vacuolation in Cos1 cells that expressed rP2X4-H286A-GFP. The bar graph on the right shows the percentage of vacuolated cells in untransfected (WT) and rP2X4-H286A-GFP–transfected Cos1 cells without or with CaM(1–4). (D) Vacuolation in Cos1 cells that expressed Lamp1-GFP without or with WT CaM or CaM(1–4). 0.4 µM thapsigargin (90 min) was applied as indicated. Thapsigargin increase vacuolation, which was enhanced by CaM but suppressed by CaM(1–4). Summary of vacuolated cells under the indicated conditions is shown on the right. Bars, 5 µm. (E–I) CaM did not alter P2X4 currents in whole-lysosome patches. Enlarged vacuoles were obtained from cells that expressed P2X4-GFP (E and F), P2X4-GFP + CaM (G), and P2X4-GFP + CaM(1–4) (H) and recorded using a pipette solution that contained 0.1 mM ATP with pH set at 7.4, without (E, G, and H) or with 10 µM Ca 2+ and 5 µM CaM (F). Left graphs show current development at −140 mV after the formation of whole-lysosome configuration; right graphs show I-V curves of currents at the indicated time points. (I) Summary of peak currents at −140 mV. All cells expressed P2X4-GFP. CaM: addition of purified CaM in the bath; rCaM and rCaM(1–4): cotransfection of rat CaM constructs. Values are means ± SEM; *, P
    Figure Legend Snippet: CaM was involved in P2X4-mediated vacuolation. (A) A CaM inhibitor, 10 µM W7 (1 h) significantly suppressed MA-induced vacuolation in Cos1 cells expressing rP2X4-GFP. Summary of vacuolated cells is shown on the right. Vacuolation was induced by either MA or Baf A1 as indicated. (B) Vacuolation of rP2X4-GFP expressing Cos1 cells was enhanced by coexpression of WT CaM but suppressed by that of CaM(1–4), a dominant-negative CaM mutant. Bar graph at right shows percentage of vacuolated cells under conditions indicated. (C) Coexpression of CaM(1–4) dramatically reduced vacuolation in Cos1 cells that expressed rP2X4-H286A-GFP. The bar graph on the right shows the percentage of vacuolated cells in untransfected (WT) and rP2X4-H286A-GFP–transfected Cos1 cells without or with CaM(1–4). (D) Vacuolation in Cos1 cells that expressed Lamp1-GFP without or with WT CaM or CaM(1–4). 0.4 µM thapsigargin (90 min) was applied as indicated. Thapsigargin increase vacuolation, which was enhanced by CaM but suppressed by CaM(1–4). Summary of vacuolated cells under the indicated conditions is shown on the right. Bars, 5 µm. (E–I) CaM did not alter P2X4 currents in whole-lysosome patches. Enlarged vacuoles were obtained from cells that expressed P2X4-GFP (E and F), P2X4-GFP + CaM (G), and P2X4-GFP + CaM(1–4) (H) and recorded using a pipette solution that contained 0.1 mM ATP with pH set at 7.4, without (E, G, and H) or with 10 µM Ca 2+ and 5 µM CaM (F). Left graphs show current development at −140 mV after the formation of whole-lysosome configuration; right graphs show I-V curves of currents at the indicated time points. (I) Summary of peak currents at −140 mV. All cells expressed P2X4-GFP. CaM: addition of purified CaM in the bath; rCaM and rCaM(1–4): cotransfection of rat CaM constructs. Values are means ± SEM; *, P

    Techniques Used: Chick Chorioallantoic Membrane Assay, Expressing, Dominant Negative Mutation, Mutagenesis, Transfection, Transferring, Purification, Cotransfection, Construct

    28) Product Images from "The effect of sphingosine-1-phosphate on colonic smooth muscle contractility: Modulation by TNBS-induced colitis"

    Article Title: The effect of sphingosine-1-phosphate on colonic smooth muscle contractility: Modulation by TNBS-induced colitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170792

    The Role of calcium and calcium sensitization pathways in S1P induced contraction in control and colitic colon. Colonic segments from control (n = 3–6) and colitic (n = 3–7) rats were equilibrated in Krebs solution for 30 min and contracted twice with KCL with consecutive washes after the maximal contraction was reached. Tissues were then preincubated with L-type calcium channel blocker (nifedipine, 1μM) or SERCA antagonist (thapsigargin, 10μM) (A) or with PKC antagonist (GF 102903x, 1μM), ROCK inhibitor (Y-27632, 10μM) or inhibitor cocktails (B) for 30 min before the addition of S1P (40μM). Data is mean ± SEM of S1P induced contraction expressed relative to the second KCl-induced contraction, * P
    Figure Legend Snippet: The Role of calcium and calcium sensitization pathways in S1P induced contraction in control and colitic colon. Colonic segments from control (n = 3–6) and colitic (n = 3–7) rats were equilibrated in Krebs solution for 30 min and contracted twice with KCL with consecutive washes after the maximal contraction was reached. Tissues were then preincubated with L-type calcium channel blocker (nifedipine, 1μM) or SERCA antagonist (thapsigargin, 10μM) (A) or with PKC antagonist (GF 102903x, 1μM), ROCK inhibitor (Y-27632, 10μM) or inhibitor cocktails (B) for 30 min before the addition of S1P (40μM). Data is mean ± SEM of S1P induced contraction expressed relative to the second KCl-induced contraction, * P

    Techniques Used:

    29) Product Images from "Inhibition of store-operated channels by carboxyamidotriazole sensitizes ovarian carcinoma cells to anti-BclxL strategies through Mcl-1 down-regulation"

    Article Title: Inhibition of store-operated channels by carboxyamidotriazole sensitizes ovarian carcinoma cells to anti-BclxL strategies through Mcl-1 down-regulation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26084

    CAI does not block IP3R calcium release but inhibits SOCE (A) Effect of CAI on IP3R and SOC. Microspectrofluorimetry using Fura-2AM probe was performed in the three carcinoma cell lines IGROV1-R10, OVCAR3 and SKOV3 cells. During exposure to 0Ca 2+ , depletion of the intracellular stores was triggered by the addition of 2 μM thapsigargin to the bathing medium. Subsequent replenishment of 2 mM Ca 2+ to the medium elicited a rise in [Ca 2+ ] i due to Ca 2+ influx through open SOC. Black tracings depict the representative changes in [Ca 2+ ] i recorded from DMSO-treated cells and grey tracings depict the representative changes in [Ca 2+ ] i recorded from cells pre-treated for 1h with 5μM CAI. (B) Means ± SEM of the peaks of thapsigargin-induced Ca 2+ release and Ca 2+ store-operated channel entry recorded from CAI pre-treated cells (black bar) or not (DMSO) (white bar) (n=3).
    Figure Legend Snippet: CAI does not block IP3R calcium release but inhibits SOCE (A) Effect of CAI on IP3R and SOC. Microspectrofluorimetry using Fura-2AM probe was performed in the three carcinoma cell lines IGROV1-R10, OVCAR3 and SKOV3 cells. During exposure to 0Ca 2+ , depletion of the intracellular stores was triggered by the addition of 2 μM thapsigargin to the bathing medium. Subsequent replenishment of 2 mM Ca 2+ to the medium elicited a rise in [Ca 2+ ] i due to Ca 2+ influx through open SOC. Black tracings depict the representative changes in [Ca 2+ ] i recorded from DMSO-treated cells and grey tracings depict the representative changes in [Ca 2+ ] i recorded from cells pre-treated for 1h with 5μM CAI. (B) Means ± SEM of the peaks of thapsigargin-induced Ca 2+ release and Ca 2+ store-operated channel entry recorded from CAI pre-treated cells (black bar) or not (DMSO) (white bar) (n=3).

    Techniques Used: Blocking Assay

    YM48583 blocks SOCE and inhibits mTORC1 targets and Mcl-1 (A) Effect of YM58483 on IP3R and SOC. Microspectrofluorimetry using Fura-2AM probe was performed in the three carcinoma cell lines IGROV1-R10, OVCAR3 and SKOV3 cells. During exposure to 0Ca 2+ , depletion of the intracellular stores was triggered by the addition of 2 μM thapsigargin to the bathing medium. Subsequent replenishment of 2 mM Ca 2+ to the medium elicited a rise in [Ca 2+ ] i due to Ca 2+ influx through open store-operated channels. Black tracings depict the representative changes in [Ca 2+ ] i recorded from DMSO treated cells and grey tracings depict the representative changes in [Ca 2+ ] i recorded from cells pre-treated 1h with 15 μM for IGROV1-R10 and OVCAR3 cells or 20 μM for SKOV3 cells (data are representative of three independent experiments). (B) Effect of YM58483 on Akt/mTORC1 pathway. Cells were treated with YM58483 (15 μM for 48h for IGROV1-R10 cells, 15 μM for 72h for OVCAR3 cells and 20 μM for 72h for SKOV3 cells). The effect of YM58483 treatment on the activation of the PI3K/Akt/mTOR pathway was analyzed by studying the protein expression of P-Akt (Ser473 and Thr308) and total Akt, P-mTORC1 (Ser2448) and P-mTORC2 (Ser2448), P-p70S6K (Thr389) and total p70S6K and P-4E-BP1 (Thr70) and total 4E-BP1 by western blot analysis. Protein levels (standardized based on actin) were determined by densitometry scanning with Image J software to generate the values shown in the bar graphs. Results are expressed as mean ± SEM. Statistical differences were analyzed with a Student t-test: * p
    Figure Legend Snippet: YM48583 blocks SOCE and inhibits mTORC1 targets and Mcl-1 (A) Effect of YM58483 on IP3R and SOC. Microspectrofluorimetry using Fura-2AM probe was performed in the three carcinoma cell lines IGROV1-R10, OVCAR3 and SKOV3 cells. During exposure to 0Ca 2+ , depletion of the intracellular stores was triggered by the addition of 2 μM thapsigargin to the bathing medium. Subsequent replenishment of 2 mM Ca 2+ to the medium elicited a rise in [Ca 2+ ] i due to Ca 2+ influx through open store-operated channels. Black tracings depict the representative changes in [Ca 2+ ] i recorded from DMSO treated cells and grey tracings depict the representative changes in [Ca 2+ ] i recorded from cells pre-treated 1h with 15 μM for IGROV1-R10 and OVCAR3 cells or 20 μM for SKOV3 cells (data are representative of three independent experiments). (B) Effect of YM58483 on Akt/mTORC1 pathway. Cells were treated with YM58483 (15 μM for 48h for IGROV1-R10 cells, 15 μM for 72h for OVCAR3 cells and 20 μM for 72h for SKOV3 cells). The effect of YM58483 treatment on the activation of the PI3K/Akt/mTOR pathway was analyzed by studying the protein expression of P-Akt (Ser473 and Thr308) and total Akt, P-mTORC1 (Ser2448) and P-mTORC2 (Ser2448), P-p70S6K (Thr389) and total p70S6K and P-4E-BP1 (Thr70) and total 4E-BP1 by western blot analysis. Protein levels (standardized based on actin) were determined by densitometry scanning with Image J software to generate the values shown in the bar graphs. Results are expressed as mean ± SEM. Statistical differences were analyzed with a Student t-test: * p

    Techniques Used: Activation Assay, Expressing, Western Blot, Software

    30) Product Images from "Mitochondrial Calcium Buffering Contributes to the Maintenance of Basal Calcium Levels in Mouse Taste Cells"

    Article Title: Mitochondrial Calcium Buffering Contributes to the Maintenance of Basal Calcium Levels in Mouse Taste Cells

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.90534.2008

    FCCP-dependent calcium elevations increase when sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps are disabled. A : FCCP-dependent calcium increases were larger after 2 μM thapsigargin (TG) application (3 min). B : measures of the peak amplitude of the FCCP-dependent calcium increase did not significantly change ( n = 24, P = 0.38) after SERCA pumps were disabled with thapsigargin. C : the duration of the FCCP-induced calcium response significantly increased after SERCA pumps were disabled ( n = 23, P = 0.00003).
    Figure Legend Snippet: FCCP-dependent calcium elevations increase when sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps are disabled. A : FCCP-dependent calcium increases were larger after 2 μM thapsigargin (TG) application (3 min). B : measures of the peak amplitude of the FCCP-dependent calcium increase did not significantly change ( n = 24, P = 0.38) after SERCA pumps were disabled with thapsigargin. C : the duration of the FCCP-induced calcium response significantly increased after SERCA pumps were disabled ( n = 23, P = 0.00003).

    Techniques Used:

    Mitochondria buffer calcium leaks across the plasma membrane and from internal stores. A : FCCP-induced calcium elevations were greatly attenuated in calcium-free external solution. Thapsigargin greatly reduced the FCCP-induced calcium elevations in calcium-free external solution. B : enlarged portions of the graph in A that correspond to FCCP applications in calcium-free external solution. The amplitude of the peak response in calcium free (see arrows) was reduced after the taste cell had been exposed to thapsigargin and most of the calcium stores had been depleted, showing that the calcium elevation measured in calcium-free external solution depends on internal calcium stores.
    Figure Legend Snippet: Mitochondria buffer calcium leaks across the plasma membrane and from internal stores. A : FCCP-induced calcium elevations were greatly attenuated in calcium-free external solution. Thapsigargin greatly reduced the FCCP-induced calcium elevations in calcium-free external solution. B : enlarged portions of the graph in A that correspond to FCCP applications in calcium-free external solution. The amplitude of the peak response in calcium free (see arrows) was reduced after the taste cell had been exposed to thapsigargin and most of the calcium stores had been depleted, showing that the calcium elevation measured in calcium-free external solution depends on internal calcium stores.

    Techniques Used:

    31) Product Images from "Contributions of SERCA pump and ryanodine-sensitive stores to presynaptic residual Ca2+"

    Article Title: Contributions of SERCA pump and ryanodine-sensitive stores to presynaptic residual Ca2+

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2010.01.004

    Effect of [Ca 2+ ] res clearance pathways on paired pulse facilitation. (A1) PPF at 50ms interpulse interval in ACSF. (A2) PPF in the same slice as A1 following a 15 min exposure to 3μM thapsigargin. (Stimulus artifacts have been truncated for clarity.)
    Figure Legend Snippet: Effect of [Ca 2+ ] res clearance pathways on paired pulse facilitation. (A1) PPF at 50ms interpulse interval in ACSF. (A2) PPF in the same slice as A1 following a 15 min exposure to 3μM thapsigargin. (Stimulus artifacts have been truncated for clarity.)

    Techniques Used:

    Filling state of presynaptic Ca 2+ stores. (A) Bath application of 3μM thapsigargin. Response to 40mM K + was used as an indication of successful Ca 2+ responses in all experiments in this Figure, but is shown only in this panel. ( n = 5). (B) Bath
    Figure Legend Snippet: Filling state of presynaptic Ca 2+ stores. (A) Bath application of 3μM thapsigargin. Response to 40mM K + was used as an indication of successful Ca 2+ responses in all experiments in this Figure, but is shown only in this panel. ( n = 5). (B) Bath

    Techniques Used:

    Block of SERCA pumps increases P1 ∫Δ F / F 0 . (A) Representative %Δ F / F 0 and ∫%Δ F / F 0 traces, (A1) ACSF. P1 (black); ΔR2 (gray), (A2) 3μM thapsigargin. P1 (black); ΔR2 (gray). (∫%Δ
    Figure Legend Snippet: Block of SERCA pumps increases P1 ∫Δ F / F 0 . (A) Representative %Δ F / F 0 and ∫%Δ F / F 0 traces, (A1) ACSF. P1 (black); ΔR2 (gray), (A2) 3μM thapsigargin. P1 (black); ΔR2 (gray). (∫%Δ

    Techniques Used: Blocking Assay

    32) Product Images from "Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits"

    Article Title: Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits

    Journal: Pflugers Archiv : European journal of physiology

    doi: 10.1007/s00424-012-1193-9

    Effects of isoproterenol in control male and female rabbit myocytes pretreated with thapsigargin
    Figure Legend Snippet: Effects of isoproterenol in control male and female rabbit myocytes pretreated with thapsigargin

    Techniques Used:

    33) Product Images from "Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus"

    Article Title: Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0941-12.2012

    Nicotinic effects depend on VGCCs and intracellular CICR. A , Time–frequency histogram shows the effect of nicotine on the sEPSC frequency in the presence of CdCl 2 (gray bar). B , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of a mixture containing the Ca 2+ channel blockers ω-agatoxin-TK, ω-conotoxin-GVIA, and nitrendipine (gray bar). C , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of the SERCA blocker thapsigargin (gray bar). D , Bar graph shows the effect of nicotine on the sEPSC frequency in slices pretreated with CdCl 2 , Ca 2+ channel blockers, thapsigargin, CPA, or ryanodine (** p
    Figure Legend Snippet: Nicotinic effects depend on VGCCs and intracellular CICR. A , Time–frequency histogram shows the effect of nicotine on the sEPSC frequency in the presence of CdCl 2 (gray bar). B , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of a mixture containing the Ca 2+ channel blockers ω-agatoxin-TK, ω-conotoxin-GVIA, and nitrendipine (gray bar). C , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of the SERCA blocker thapsigargin (gray bar). D , Bar graph shows the effect of nicotine on the sEPSC frequency in slices pretreated with CdCl 2 , Ca 2+ channel blockers, thapsigargin, CPA, or ryanodine (** p

    Techniques Used:

    34) Product Images from "Synaptic contributions to cochlear outer hair cell Ca2+ homeostasis"

    Article Title: Synaptic contributions to cochlear outer hair cell Ca2+ homeostasis

    Journal: bioRxiv

    doi: 10.1101/2020.08.02.233205

    Modulation of afferent Ca 2+ influx through VGCC by ryanodine and sorcin. A) Mean traces of the Ca 2+ transients obtained with 300 msec depolarization to +20 mV before (top) and during perfusion of Thapsigargin (bottom). B) Average peak Ca 2+ level (ΔF) for step depolarizations as in A). C) Mean traces of the Ca 2+ transients during with steps to +20 mV, using intracellular solutions containing DMSO or Ryanodine 1 µM. D) Average peak Ca 2+ signal (ΔF/F 0 ) for experiments in C), and also with an antagonistic concentration of ryanodine (Ry) (100 μM) and dantrolene (30 μM). Wilcoxon signed-rank test, * p
    Figure Legend Snippet: Modulation of afferent Ca 2+ influx through VGCC by ryanodine and sorcin. A) Mean traces of the Ca 2+ transients obtained with 300 msec depolarization to +20 mV before (top) and during perfusion of Thapsigargin (bottom). B) Average peak Ca 2+ level (ΔF) for step depolarizations as in A). C) Mean traces of the Ca 2+ transients during with steps to +20 mV, using intracellular solutions containing DMSO or Ryanodine 1 µM. D) Average peak Ca 2+ signal (ΔF/F 0 ) for experiments in C), and also with an antagonistic concentration of ryanodine (Ry) (100 μM) and dantrolene (30 μM). Wilcoxon signed-rank test, * p

    Techniques Used: Concentration Assay

    Efferent Ca 2+ signals are modulated by cisternal ATPases, but not ryanodine receptors. A) Mean synaptic responses (black) and Ca 2+ transients (red) during 300 msec electrical stimulation at 20,40 and 80 Hz before and after perfusion of Thapsigargin. B) Peak of Ca 2+ transients (as ΔF), C) Charge of synaptic responses and D) duration of Ca 2+ transients (as full width at half maximum, FWHM). Inset: Baseline fluorescence signal before and after perfusion of Thapsigargin. E) Synaptic currents (mean, black) and Ca 2+ transients (red) obtained during efferent fibers stimulation (300 msec, 80 Hz), using an intracellular solution containing vehicle (DMSO), RyR blockers (Ryanodine 100 µM and Dantrolene 30 µM) and a RyR agonist (Ryanodine 1 µM). F) Baseline fluorescence for each condition. G) Duration (FWHM) and H) maximal fluorescence signal (as ΔF/F 0 ) for each intracellular solution. Bar plots are mean ± SEM. Wilcoxon signed-rank test, * p
    Figure Legend Snippet: Efferent Ca 2+ signals are modulated by cisternal ATPases, but not ryanodine receptors. A) Mean synaptic responses (black) and Ca 2+ transients (red) during 300 msec electrical stimulation at 20,40 and 80 Hz before and after perfusion of Thapsigargin. B) Peak of Ca 2+ transients (as ΔF), C) Charge of synaptic responses and D) duration of Ca 2+ transients (as full width at half maximum, FWHM). Inset: Baseline fluorescence signal before and after perfusion of Thapsigargin. E) Synaptic currents (mean, black) and Ca 2+ transients (red) obtained during efferent fibers stimulation (300 msec, 80 Hz), using an intracellular solution containing vehicle (DMSO), RyR blockers (Ryanodine 100 µM and Dantrolene 30 µM) and a RyR agonist (Ryanodine 1 µM). F) Baseline fluorescence for each condition. G) Duration (FWHM) and H) maximal fluorescence signal (as ΔF/F 0 ) for each intracellular solution. Bar plots are mean ± SEM. Wilcoxon signed-rank test, * p

    Techniques Used: Fluorescence

    35) Product Images from "Prefrontal Cortex Stimulation Induces 2-Arachidonoyl-Glycerol-Mediated Suppression of Excitation in Dopamine Neurons"

    Article Title: Prefrontal Cortex Stimulation Induces 2-Arachidonoyl-Glycerol-Mediated Suppression of Excitation in Dopamine Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3502-04.2004

    Contribution of intracellular calcium, glutamate, and D 2 DA receptors to 2-AG-induced effects. A , Magnitude of EPSC amplitude after the train for all conditions [thapsigargin (thapsi); ruthenium red (RR); ryanodine (ryano); n = 7] plotted as the percentage of baseline before the train (dotted line). B , Magnitude of EPSC amplitude after the train for all conditions (AP5, n = 7; MCPG, n = 7; CPCCOEt, n = 6; MPEP, n = 6; eticlopride, n = 7) plotted as the percentage of baseline before the train (dotted line). Representative traces for each condition from a single experiment are shown. The EPSC recorded after the train is superimposed in light gray for comparison. Calibration: 20 msec, 100 pA.
    Figure Legend Snippet: Contribution of intracellular calcium, glutamate, and D 2 DA receptors to 2-AG-induced effects. A , Magnitude of EPSC amplitude after the train for all conditions [thapsigargin (thapsi); ruthenium red (RR); ryanodine (ryano); n = 7] plotted as the percentage of baseline before the train (dotted line). B , Magnitude of EPSC amplitude after the train for all conditions (AP5, n = 7; MCPG, n = 7; CPCCOEt, n = 6; MPEP, n = 6; eticlopride, n = 7) plotted as the percentage of baseline before the train (dotted line). Representative traces for each condition from a single experiment are shown. The EPSC recorded after the train is superimposed in light gray for comparison. Calibration: 20 msec, 100 pA.

    Techniques Used:

    36) Product Images from "CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts"

    Article Title: CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2605-y

    Both KN-62 and KB-R7943 ameliorated heart injury in Ca 2+ paradox, but combined treatment with both ryanodine and thapsigargin did not. a Representative heart slice TTC staining. Scale bar, 0.5 cm. b , c Grouped results of myocardial injury area and LDH release in the coronary effluent, respectively. d Grouped results of caspase-3 activity. e Representative blots of cytochrome c in mitochondrial and cytosolic fraction and grouped results of densitometric analysis. GAPDH and COX IV served as a loading controls. The values are expressed as the percentages of the control values. Each bar represents the mean ± SEM; n = 6 rats in each group. Western blots were performed in five independent biological experiments and for three technical replicates per sample. ##, ** P
    Figure Legend Snippet: Both KN-62 and KB-R7943 ameliorated heart injury in Ca 2+ paradox, but combined treatment with both ryanodine and thapsigargin did not. a Representative heart slice TTC staining. Scale bar, 0.5 cm. b , c Grouped results of myocardial injury area and LDH release in the coronary effluent, respectively. d Grouped results of caspase-3 activity. e Representative blots of cytochrome c in mitochondrial and cytosolic fraction and grouped results of densitometric analysis. GAPDH and COX IV served as a loading controls. The values are expressed as the percentages of the control values. Each bar represents the mean ± SEM; n = 6 rats in each group. Western blots were performed in five independent biological experiments and for three technical replicates per sample. ##, ** P

    Techniques Used: Staining, Activity Assay, Western Blot

    37) Product Images from "Ca2+ signaling behaviours of intramuscular interstitial cells of Cajal in the murine colon"

    Article Title: Ca2+ signaling behaviours of intramuscular interstitial cells of Cajal in the murine colon

    Journal: The Journal of physiology

    doi: 10.1113/JP278036

    Ca 2+ transients in ICC-IM depend upon functional intracellular Ca 2+ stores and Ca 2+ influx. Ai-ii Representative STMs of Ca 2+ transient firing in a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 10 μM thapsigargin (ii). Bi-ii Representative STMs of Ca 2+ transient firing within a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 10 μM CPA (ii). Ci-ii Representative STMs of Ca 2+ transient firing within a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 0 mM [Ca 2+ ] o solution for 10 mins (ii). Di-iv Summary data showing the effect of 0 mM [Ca 2+ ] o solution on ICC-IM Ca 2+ transient frequency (i), amplitude (ii), duration (iii) and spatial spread (iv) after 2, 4, 6, 8 and 10 mins (c=19, n=5).
    Figure Legend Snippet: Ca 2+ transients in ICC-IM depend upon functional intracellular Ca 2+ stores and Ca 2+ influx. Ai-ii Representative STMs of Ca 2+ transient firing in a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 10 μM thapsigargin (ii). Bi-ii Representative STMs of Ca 2+ transient firing within a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 10 μM CPA (ii). Ci-ii Representative STMs of Ca 2+ transient firing within a single colonic ICC-IM recorded in situ during control conditions (i) and after incubation with 0 mM [Ca 2+ ] o solution for 10 mins (ii). Di-iv Summary data showing the effect of 0 mM [Ca 2+ ] o solution on ICC-IM Ca 2+ transient frequency (i), amplitude (ii), duration (iii) and spatial spread (iv) after 2, 4, 6, 8 and 10 mins (c=19, n=5).

    Techniques Used: Immunocytochemistry, Functional Assay, In Situ, Incubation

    38) Product Images from "Pregnancy associated plasma protein-aa regulates endoplasmic reticulum-mitochondria associations"

    Article Title: Pregnancy associated plasma protein-aa regulates endoplasmic reticulum-mitochondria associations

    Journal: bioRxiv

    doi: 10.1101/2020.06.09.142505

    pappaa p170 hair cells are more sensitive to disruption in ER-mitochondria calcium signaling. (A) Schematic of calcium channel modulators. Thapsigargin inhibits calcium uptake by the ER by blocking the SERCA pump. Adenophostin A stimulates calcium release through activation of the IP3Rs. (B) Representative images of brn3c:mGFP labeled hair cells from vehicle, 10 µM Thapsigargin, or 10 µM Adenophostin A treated larvae. (C) Mean percentage of surviving hair cells following a 1-hour treatment with either Thapsigargin or Adenosphostin A at 5 dpf. To calculate hair cell survival percentage, hair cell number 4 hours post-drug treatment was normalized to mean hair cell number in vehicle treated larvae of the same genotype. * p
    Figure Legend Snippet: pappaa p170 hair cells are more sensitive to disruption in ER-mitochondria calcium signaling. (A) Schematic of calcium channel modulators. Thapsigargin inhibits calcium uptake by the ER by blocking the SERCA pump. Adenophostin A stimulates calcium release through activation of the IP3Rs. (B) Representative images of brn3c:mGFP labeled hair cells from vehicle, 10 µM Thapsigargin, or 10 µM Adenophostin A treated larvae. (C) Mean percentage of surviving hair cells following a 1-hour treatment with either Thapsigargin or Adenosphostin A at 5 dpf. To calculate hair cell survival percentage, hair cell number 4 hours post-drug treatment was normalized to mean hair cell number in vehicle treated larvae of the same genotype. * p

    Techniques Used: Blocking Assay, Activation Assay, Labeling

    39) Product Images from "A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli"

    Article Title: A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli

    Journal: bioRxiv

    doi: 10.1101/660589

    Taste-evoked Ca 2+ release in IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. Representative data related to Figure 4 . Open columns represent the time that the taste stimulus is presented. The application of Ca 2+ free Tyrode’s is indicated by the dashed lines. The stimulus presented during this time is also in Ca 2+ free Tyrode’s. The gray hatched columns represent the application of either thapsigargin (Thap) or U73122, both of which are irreversible inhibitors. A) Bitter-evoked taste responses (5mM Den) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B) as well as the PLC blocker U73122 (C). D) Responses to sweet stimuli (20mM sucralose, Sucr) persist in Ca 2+ -free and are abolished by thapsigargin (E) and U73122 (F). G) Umami stimuli (10mM MPG) persist in Ca 2+ -free and were abolished by thapsigargin (H) and U73122 (I).
    Figure Legend Snippet: Taste-evoked Ca 2+ release in IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. Representative data related to Figure 4 . Open columns represent the time that the taste stimulus is presented. The application of Ca 2+ free Tyrode’s is indicated by the dashed lines. The stimulus presented during this time is also in Ca 2+ free Tyrode’s. The gray hatched columns represent the application of either thapsigargin (Thap) or U73122, both of which are irreversible inhibitors. A) Bitter-evoked taste responses (5mM Den) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B) as well as the PLC blocker U73122 (C). D) Responses to sweet stimuli (20mM sucralose, Sucr) persist in Ca 2+ -free and are abolished by thapsigargin (E) and U73122 (F). G) Umami stimuli (10mM MPG) persist in Ca 2+ -free and were abolished by thapsigargin (H) and U73122 (I).

    Techniques Used: Mouse Assay, Planar Chromatography, Activity Assay

    Taste-evoked Ca 2+ release in BR cells from IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. A) Bitter-evoked taste responses (5mM Denatonium, n=6) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B, n=7) as well as the PLC blocker U73122 (C, n=7). D) Responses to sweet stimuli (50mM sucrose, n=6) persist in Ca 2+ -free and are abolished by thapsigargin (E, n=4) and U73122 (F, n=6). G) Umami stimuli (10mM MPG) persist in Ca 2+ -free (n=7) and were abolished by thapsigargin (H, n=4) and U73122 (I, n=4). Representative data for each experiment are shown in Figure S3 . Comparisons of the response amplitudes for each taste stimulus found no significant differences in the control responses between experiments (One way ANOVA, Den, p=0.933; Suc, p=0.623; MPG, p=0.134).
    Figure Legend Snippet: Taste-evoked Ca 2+ release in BR cells from IP 3 R3-KO mice is dependent upon PLC activity and Ca 2+ release from internal stores. A) Bitter-evoked taste responses (5mM Denatonium, n=6) persist in the absence of extracellular calcium (Ca 2+ -free) and are abolished by the SERCA pump inhibitor thapsigargin (B, n=7) as well as the PLC blocker U73122 (C, n=7). D) Responses to sweet stimuli (50mM sucrose, n=6) persist in Ca 2+ -free and are abolished by thapsigargin (E, n=4) and U73122 (F, n=6). G) Umami stimuli (10mM MPG) persist in Ca 2+ -free (n=7) and were abolished by thapsigargin (H, n=4) and U73122 (I, n=4). Representative data for each experiment are shown in Figure S3 . Comparisons of the response amplitudes for each taste stimulus found no significant differences in the control responses between experiments (One way ANOVA, Den, p=0.933; Suc, p=0.623; MPG, p=0.134).

    Techniques Used: Mouse Assay, Planar Chromatography, Activity Assay

    40) Product Images from "Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells"

    Article Title: Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-015-0107-9

    Effect of the TGR5 ligands on [Ca 2+ ] i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca 2+ buffer ( a , b ) and thapsigargin (1 μM) to deplete intracellular Ca 2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca 2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c , d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na + buffer which increased [Ca 2+ ] i and this response was potentiated in the presence of GPBAR-A (30 μM). b , d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P
    Figure Legend Snippet: Effect of the TGR5 ligands on [Ca 2+ ] i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca 2+ buffer ( a , b ) and thapsigargin (1 μM) to deplete intracellular Ca 2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca 2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c , d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na + buffer which increased [Ca 2+ ] i and this response was potentiated in the presence of GPBAR-A (30 μM). b , d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P

    Techniques Used: Incubation, Fluorescence

    Effect of P2 receptor inhibitors on intracellular Ca 2+ responses induced by CDCA in Capan-1 cells. a and b Representative recordings of [Ca 2+ ] i transients in Capan-1 cells with or without P2 receptors inhibitors (standing bath). Cell were incubated with a mix of P2R inhibitors: PPADS (250 μM), suramin (250 μM), and 10 μM of AZ 10606120 and 10 μM of A438079 for 25 min. a , c The presence of P2R antagonists markedly inhibited [Ca 2+ ] i response induced by ATP (100 μM), but had no effect on Thapsigargin (1 μM) induced Ca 2+ response. b , d Incubation cells with P2R antagonists inhibited [Ca 2+ ] i transient induced by 0.3 mM CDCA but not by Thapsigargin (1 μM). c , d Change in [Ca 2+ ] i above baseline are given as mean values ± SEM of 7–15 cells per each independent experiment (n). Arrows indicate the time of adding the stimuli. *= P
    Figure Legend Snippet: Effect of P2 receptor inhibitors on intracellular Ca 2+ responses induced by CDCA in Capan-1 cells. a and b Representative recordings of [Ca 2+ ] i transients in Capan-1 cells with or without P2 receptors inhibitors (standing bath). Cell were incubated with a mix of P2R inhibitors: PPADS (250 μM), suramin (250 μM), and 10 μM of AZ 10606120 and 10 μM of A438079 for 25 min. a , c The presence of P2R antagonists markedly inhibited [Ca 2+ ] i response induced by ATP (100 μM), but had no effect on Thapsigargin (1 μM) induced Ca 2+ response. b , d Incubation cells with P2R antagonists inhibited [Ca 2+ ] i transient induced by 0.3 mM CDCA but not by Thapsigargin (1 μM). c , d Change in [Ca 2+ ] i above baseline are given as mean values ± SEM of 7–15 cells per each independent experiment (n). Arrows indicate the time of adding the stimuli. *= P

    Techniques Used: Incubation

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    Article Snippet: Thapsigargin was purchased from Tocris Bioscience (Minneapolis, MN).

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    Article Snippet: Thapsigargin was obtained from Tocris Biosciences (Bristol, UK).

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    Tocris inhibitors thapsigargin
    (A) MDA-MB-231 cells expressing GFP-E-Syt1 and mCherry-ORP3 were treated with <t>thapsigargin</t> (1 µM) and imaged live for 10 min. Panels show stills before and after thapsigargin treatment. Bar = 10 µm. (B) MDA-MB-231 cells expressing GFP-ORP3 and mCherry-STIM1 were treated with EGF (100 ng/ml) and imaged for 30 min. Panels show stills before and after EGF treatment. Bar = 10 µm (B’) Quantitation of ORP3 fluorescence at the PM, after EGF (100ng/ml) treatment. Data were collected from 5 cells. (C) Representative stills from movies of live cells expressing either GFP-ORP3 or GFP-ORP3Δ PH , as indicated. Third panel shows cells expressing WT GFP-ORP3 pretreated with wortmanin (100nM). All three groups of cells were treated with thapsigargin (1µM) and imaged live. Images show that recruitment of ORP3 to the PM is dependent upon its PH domain, but is not inhibited by wortmanin. Bar = 10 µm (D) Quantitation of percentage of ORP3 puncta at the PM after thapsigargin treatment for experiments shown in panel C. Data were collected from 5 cells per group. (E) Live cells co-expressing the PI4P probe GFP-P4M2X and mCherry-ORP3 were pre-treated with the PI4KIIIα inhibitor GSK-A1 (30 nM) for 10mins and then imaged for 2.5 mins. After the loss of GFP-P4M2X signal, cells were treated with thapsigargin and imaged for an additional 30 mins. The changes in ORP3 fluorescence at the PM were quantified and graphed. (F) MDA-MB-231 cells expressing GFP-ORP3Δ ORD mutant were treated with GSK-A1 for 20 mins and then treated with thapsigargin. Representative images show cells before and after thapsigargin treatment.
    Inhibitors Thapsigargin, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hyperthermia increases ATP-tumor killing activity by enhancing P2X7 pore formation independently of Ca 2+ influx and pannexin/connexin interaction (A) P2X7 functionality upon ATP/hyperthermia treatment measured by etidium bromide (EtBr) uptake. Cells were left untreated or treated with ATP (1 mM) for 15 min at 37°C or 40°C, followed by whole cell fluorescence measurement (AUF), as described in Material and Methods. (B) Cells were pre-incubated with BAPTA-AM, <t>thapsigargin</t> (TG) or Carbenoxolone (CBX) prior to heat-ATP pulse treatment, followed by western blot analysis of AKT/PRAS40/mTOR signaling pathway. Cells treated with media served as control. (C) NC (negative control) and P2X7 KD (P2X7-deficient) cells were exposed to ATP for 15 min at 37°C or 40°C and extracellular and intracellular adenine nucleotide levels were determined by HPLC. *p
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    (A) MDA-MB-231 cells expressing GFP-E-Syt1 and mCherry-ORP3 were treated with thapsigargin (1 µM) and imaged live for 10 min. Panels show stills before and after thapsigargin treatment. Bar = 10 µm. (B) MDA-MB-231 cells expressing GFP-ORP3 and mCherry-STIM1 were treated with EGF (100 ng/ml) and imaged for 30 min. Panels show stills before and after EGF treatment. Bar = 10 µm (B’) Quantitation of ORP3 fluorescence at the PM, after EGF (100ng/ml) treatment. Data were collected from 5 cells. (C) Representative stills from movies of live cells expressing either GFP-ORP3 or GFP-ORP3Δ PH , as indicated. Third panel shows cells expressing WT GFP-ORP3 pretreated with wortmanin (100nM). All three groups of cells were treated with thapsigargin (1µM) and imaged live. Images show that recruitment of ORP3 to the PM is dependent upon its PH domain, but is not inhibited by wortmanin. Bar = 10 µm (D) Quantitation of percentage of ORP3 puncta at the PM after thapsigargin treatment for experiments shown in panel C. Data were collected from 5 cells per group. (E) Live cells co-expressing the PI4P probe GFP-P4M2X and mCherry-ORP3 were pre-treated with the PI4KIIIα inhibitor GSK-A1 (30 nM) for 10mins and then imaged for 2.5 mins. After the loss of GFP-P4M2X signal, cells were treated with thapsigargin and imaged for an additional 30 mins. The changes in ORP3 fluorescence at the PM were quantified and graphed. (F) MDA-MB-231 cells expressing GFP-ORP3Δ ORD mutant were treated with GSK-A1 for 20 mins and then treated with thapsigargin. Representative images show cells before and after thapsigargin treatment.

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: (A) MDA-MB-231 cells expressing GFP-E-Syt1 and mCherry-ORP3 were treated with thapsigargin (1 µM) and imaged live for 10 min. Panels show stills before and after thapsigargin treatment. Bar = 10 µm. (B) MDA-MB-231 cells expressing GFP-ORP3 and mCherry-STIM1 were treated with EGF (100 ng/ml) and imaged for 30 min. Panels show stills before and after EGF treatment. Bar = 10 µm (B’) Quantitation of ORP3 fluorescence at the PM, after EGF (100ng/ml) treatment. Data were collected from 5 cells. (C) Representative stills from movies of live cells expressing either GFP-ORP3 or GFP-ORP3Δ PH , as indicated. Third panel shows cells expressing WT GFP-ORP3 pretreated with wortmanin (100nM). All three groups of cells were treated with thapsigargin (1µM) and imaged live. Images show that recruitment of ORP3 to the PM is dependent upon its PH domain, but is not inhibited by wortmanin. Bar = 10 µm (D) Quantitation of percentage of ORP3 puncta at the PM after thapsigargin treatment for experiments shown in panel C. Data were collected from 5 cells per group. (E) Live cells co-expressing the PI4P probe GFP-P4M2X and mCherry-ORP3 were pre-treated with the PI4KIIIα inhibitor GSK-A1 (30 nM) for 10mins and then imaged for 2.5 mins. After the loss of GFP-P4M2X signal, cells were treated with thapsigargin and imaged for an additional 30 mins. The changes in ORP3 fluorescence at the PM were quantified and graphed. (F) MDA-MB-231 cells expressing GFP-ORP3Δ ORD mutant were treated with GSK-A1 for 20 mins and then treated with thapsigargin. Representative images show cells before and after thapsigargin treatment.

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Multiple Displacement Amplification, Expressing, Quantitation Assay, Fluorescence, Mutagenesis

    (A) Predictive model of IQSec1, generated using Raptor-X. SEC7-PH domain tandem (based on the existing crystal structure) is at the right, predicted helices are numbered 1-4. Arrow indicates position of the IQ motif. ORP3 binding loop is indicated by black arrows. (B) Cells co-expressing GFP-ORP3 with either mCherry WT IQSec1 or IQSec1Δ 152-212 were lysed and immunoprecipitated with anti-mCherry antibody, then probed for GFP-ORP3. (C) Domain organization of ORP3 and ORP3 truncation mutants; PH (blue) = PH domain, FFAT (black) = two phenylalanines in an acidic tract, ORD (yellow) = OSPB-related domain. (D) Lysates of cells co-expressing WT mCherry-IQSec1 and various mutants of GFP-ORP3 were immunoprecipitated with anti-mCherry and probed for GFP. (E) Cells expressing equivalent amounts of ORP3 and IQSec1 were transfected with increasing amounts of vector encoding VAP-A. Cells were then lysed, IQSec1 was immunoprecipitated and the blot probed for co-precipitating ORP3. (F) Quantification of ORP3 co-precipitation with IQSec1 in the presence of increasing amounts of VAPA. Data were compiled from two independent experiments. (G) MDA-MB-231 cells expressing the PI4P probe GFP-P4M2X and mCherry-ORP3 1-555 (ΔORD) were imaged live. Images show stills before after treatment with thapsigargin (1µM). The changes in PM fluorescence for GFP-P4M2X/mCherry-ORP3, were quantified and shown in (H). (H) Data were collected from 7 cells in each group.

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: (A) Predictive model of IQSec1, generated using Raptor-X. SEC7-PH domain tandem (based on the existing crystal structure) is at the right, predicted helices are numbered 1-4. Arrow indicates position of the IQ motif. ORP3 binding loop is indicated by black arrows. (B) Cells co-expressing GFP-ORP3 with either mCherry WT IQSec1 or IQSec1Δ 152-212 were lysed and immunoprecipitated with anti-mCherry antibody, then probed for GFP-ORP3. (C) Domain organization of ORP3 and ORP3 truncation mutants; PH (blue) = PH domain, FFAT (black) = two phenylalanines in an acidic tract, ORD (yellow) = OSPB-related domain. (D) Lysates of cells co-expressing WT mCherry-IQSec1 and various mutants of GFP-ORP3 were immunoprecipitated with anti-mCherry and probed for GFP. (E) Cells expressing equivalent amounts of ORP3 and IQSec1 were transfected with increasing amounts of vector encoding VAP-A. Cells were then lysed, IQSec1 was immunoprecipitated and the blot probed for co-precipitating ORP3. (F) Quantification of ORP3 co-precipitation with IQSec1 in the presence of increasing amounts of VAPA. Data were compiled from two independent experiments. (G) MDA-MB-231 cells expressing the PI4P probe GFP-P4M2X and mCherry-ORP3 1-555 (ΔORD) were imaged live. Images show stills before after treatment with thapsigargin (1µM). The changes in PM fluorescence for GFP-P4M2X/mCherry-ORP3, were quantified and shown in (H). (H) Data were collected from 7 cells in each group.

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Generated, Binding Assay, Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Multiple Displacement Amplification, Fluorescence

    ORP3 extracts PI4P from the plasma membrane (A-D) MDA-MB-231 cells expressing the PI4P probe, GFP-P4M2X and either mCherry alone (A) or mCherry-ORP3 (C) were imaged live. Images show stills before (Frame1) and after (Frame75) treatment with thapsigargin (1µM) for 30 mins. The changes in PM fluorescence for GFP-P4M2X/mCherry and GFP-P4M2X/mCherry-ORP3, were quantified and presented in (B) and (D) respectively. Data were compiled from 5 cells per group. (E) Cells expressing GFP-Paxillin were treated with GSK-A1 to deplete PM PI4P and focal adhesion size was measured before and after treatment. Data were collected from 7 cells per group (F) Quantification of cell migration out of spheroids from control and GSK-A1-treated cells after 22hrs in 3D collagen gels. Data were collected from 6 spheroids for each group (G) Quantification of PC precipitated with GFP and ORP3 protein before and after thapsigargin treatment. (H) FA disassembly rates in cells expressing GFP-paxillin. ORP3 depleted cells were transfected with constructs encoding WT ORP3 or Δ FFAT or Δ ORD mutants of ORP3 and FA turnover was measured as described in Figure. 1 . N for control = 351, ORP3 KD = 306, ORP3 KD+ Rescue WT = 304, ORP3 KD+ Rescue Δ FFAT = 373, ORP3 KD+ Rescue Δ ORD = 286 focal adhesions. Data were compiled from 7 cells per group. Note that the disassembly rates for ORP3 KD+ Rescue WT are significantly higher than WT ORP3 KD+ Rescue Δ ORD (I) FA disassembly rates were measured in ORP3 depleted cells expressing the rapid cycling T161A mutant of Arf5. Two separate hairpins were used to knockdown ORP3 for this experiment. N for control = 307, ORP3 KD-1 = 383, ORP3 KD-1 + ARF5 RC = 231, ORP3 KD-2= 296 and ORP3 KD-2 + ARF5 RC = 120. Data were compiled from 5 cells per group.

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: ORP3 extracts PI4P from the plasma membrane (A-D) MDA-MB-231 cells expressing the PI4P probe, GFP-P4M2X and either mCherry alone (A) or mCherry-ORP3 (C) were imaged live. Images show stills before (Frame1) and after (Frame75) treatment with thapsigargin (1µM) for 30 mins. The changes in PM fluorescence for GFP-P4M2X/mCherry and GFP-P4M2X/mCherry-ORP3, were quantified and presented in (B) and (D) respectively. Data were compiled from 5 cells per group. (E) Cells expressing GFP-Paxillin were treated with GSK-A1 to deplete PM PI4P and focal adhesion size was measured before and after treatment. Data were collected from 7 cells per group (F) Quantification of cell migration out of spheroids from control and GSK-A1-treated cells after 22hrs in 3D collagen gels. Data were collected from 6 spheroids for each group (G) Quantification of PC precipitated with GFP and ORP3 protein before and after thapsigargin treatment. (H) FA disassembly rates in cells expressing GFP-paxillin. ORP3 depleted cells were transfected with constructs encoding WT ORP3 or Δ FFAT or Δ ORD mutants of ORP3 and FA turnover was measured as described in Figure. 1 . N for control = 351, ORP3 KD = 306, ORP3 KD+ Rescue WT = 304, ORP3 KD+ Rescue Δ FFAT = 373, ORP3 KD+ Rescue Δ ORD = 286 focal adhesions. Data were compiled from 7 cells per group. Note that the disassembly rates for ORP3 KD+ Rescue WT are significantly higher than WT ORP3 KD+ Rescue Δ ORD (I) FA disassembly rates were measured in ORP3 depleted cells expressing the rapid cycling T161A mutant of Arf5. Two separate hairpins were used to knockdown ORP3 for this experiment. N for control = 307, ORP3 KD-1 = 383, ORP3 KD-1 + ARF5 RC = 231, ORP3 KD-2= 296 and ORP3 KD-2 + ARF5 RC = 120. Data were compiled from 5 cells per group.

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Multiple Displacement Amplification, Expressing, Fluorescence, Migration, Transfection, Construct, Mutagenesis

    (A) MDA-MB-231 cells expressing GFP-STIM1 and DsRed-Paxillin were imaged live. A single frame from a movie ( Movie S3 ) showing bright STIM1 puncta near disassembling FAs, indicated by arrows, Bar = 2.5 µm. (B) mCherry-STIM1 was expressed in control, IQSec1-depleted or ARF5-depleted cells. Cells were then imaged live before and after thapsigargin (1µM) treatment for 10 min. Representative stills in (B) show a robust recruitment of STIM1 after thapsigargin treatment (Bar = 10µm), quantified in (C). Data shown in (C) represent the percentage of STIM1 puncta making contact with the PM after thapsigargin treatment, normalized to amount of pre-existing PM before thapsigargin treatment. Data were collected from 5 cells per group. (D) Lysates from cells co-expressing GFP-PTPN13 and either unfused mcherry or mCherry-IQSec1 or mCherry-IQSec1Δ PDZ , a mutant lacking the PDZ binding site were immunoprecipitated using anti-mCherry antibody and probed using anti-GFP antibody. PTPN13P co-precipitates with IQSec1 but not with mcherry or with IQSec1 mutant lacking the PDZ binding region. (D’) Lysates from cells co-expressing mCherry-IQSec1 and either unfused GFP or GFP-Sty1,2, or 3 (left) were immunoprecipitated with GFP-TRAP beads (Chromotek) and probed for GFP and mCherry (right). IQSec1 co-precipitates with GFP-E-Syt3 but not GFP alone. (E) Quantification of FA size from control or ORP3 depleted HT1080cells, using vinculin as a marker for FA. N for control = 758, ORP3 KD-hairpin1 = 853 ORP3 KD-hairpin2=617 adhesions. Data were collected from 10 cells

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: (A) MDA-MB-231 cells expressing GFP-STIM1 and DsRed-Paxillin were imaged live. A single frame from a movie ( Movie S3 ) showing bright STIM1 puncta near disassembling FAs, indicated by arrows, Bar = 2.5 µm. (B) mCherry-STIM1 was expressed in control, IQSec1-depleted or ARF5-depleted cells. Cells were then imaged live before and after thapsigargin (1µM) treatment for 10 min. Representative stills in (B) show a robust recruitment of STIM1 after thapsigargin treatment (Bar = 10µm), quantified in (C). Data shown in (C) represent the percentage of STIM1 puncta making contact with the PM after thapsigargin treatment, normalized to amount of pre-existing PM before thapsigargin treatment. Data were collected from 5 cells per group. (D) Lysates from cells co-expressing GFP-PTPN13 and either unfused mcherry or mCherry-IQSec1 or mCherry-IQSec1Δ PDZ , a mutant lacking the PDZ binding site were immunoprecipitated using anti-mCherry antibody and probed using anti-GFP antibody. PTPN13P co-precipitates with IQSec1 but not with mcherry or with IQSec1 mutant lacking the PDZ binding region. (D’) Lysates from cells co-expressing mCherry-IQSec1 and either unfused GFP or GFP-Sty1,2, or 3 (left) were immunoprecipitated with GFP-TRAP beads (Chromotek) and probed for GFP and mCherry (right). IQSec1 co-precipitates with GFP-E-Syt3 but not GFP alone. (E) Quantification of FA size from control or ORP3 depleted HT1080cells, using vinculin as a marker for FA. N for control = 758, ORP3 KD-hairpin1 = 853 ORP3 KD-hairpin2=617 adhesions. Data were collected from 10 cells

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Multiple Displacement Amplification, Expressing, Mutagenesis, Binding Assay, Immunoprecipitation, Marker

    ORP3 is recruited to STIM1-positive ER/PM contacts in response to calcium influx (A) Still images from live cells (Movie S5) co-expressing GFP-ORP3 and mCherry-STIM1, before and after thapsigargin (1µM) treatment. Bar = 10µm. (B) Quantitation of ORP3 colocalization with STIM1 in response to thapsigargin, added 2.5 mins after the start of the movie. Data were collected from 5 cells. (C) Time series from MDA-MB-231cell expressing GFP-ORP3, imaged before and after thapsigargin (1µM) treatment (arrow indicates time of addition) showing robust recruitment of ORP3 to the PM after treatment with thapsigargin. Bar = 5µm (D) Quantitation of ORP3 fluorescence at the PM, after thapsigargin treatment. Data were collected from 5 cells. (E) Live cells expressing GFP-ORP3 were pre-treated with either DMSO or with BTP2 (25µM) for 2 hr, then treated with thapsigargin (1µM) for 5 min. Recruitment ORP3 to the PM is blocked by BTP2. Bar = 10µm (F) Percentage of GFP-ORP3 engaged with the PM after thapsigargin treatment in the presence and absence of BTP2. Data were collected from 5 cells per group. (G) Percentage of GFP-ORP3 localized to the PM after thapsigargin treatment in control vs. STIM1 depleted cells. Data were collected from 6 cells per group. (H) Interaction between IQSec1 and ORP3 is stimulated by SOCE and inhibited when calcium influx is blocked by BTP2. Note the shift in ORP3 mobility upon thapsigargin treatment. This experiment was repeated twice and a similar trend was noted.

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: ORP3 is recruited to STIM1-positive ER/PM contacts in response to calcium influx (A) Still images from live cells (Movie S5) co-expressing GFP-ORP3 and mCherry-STIM1, before and after thapsigargin (1µM) treatment. Bar = 10µm. (B) Quantitation of ORP3 colocalization with STIM1 in response to thapsigargin, added 2.5 mins after the start of the movie. Data were collected from 5 cells. (C) Time series from MDA-MB-231cell expressing GFP-ORP3, imaged before and after thapsigargin (1µM) treatment (arrow indicates time of addition) showing robust recruitment of ORP3 to the PM after treatment with thapsigargin. Bar = 5µm (D) Quantitation of ORP3 fluorescence at the PM, after thapsigargin treatment. Data were collected from 5 cells. (E) Live cells expressing GFP-ORP3 were pre-treated with either DMSO or with BTP2 (25µM) for 2 hr, then treated with thapsigargin (1µM) for 5 min. Recruitment ORP3 to the PM is blocked by BTP2. Bar = 10µm (F) Percentage of GFP-ORP3 engaged with the PM after thapsigargin treatment in the presence and absence of BTP2. Data were collected from 5 cells per group. (G) Percentage of GFP-ORP3 localized to the PM after thapsigargin treatment in control vs. STIM1 depleted cells. Data were collected from 6 cells per group. (H) Interaction between IQSec1 and ORP3 is stimulated by SOCE and inhibited when calcium influx is blocked by BTP2. Note the shift in ORP3 mobility upon thapsigargin treatment. This experiment was repeated twice and a similar trend was noted.

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Expressing, Quantitation Assay, Multiple Displacement Amplification, Fluorescence

    Phosphoregulation of ORP3 recruitment to the PM (A) MDA-MB-231cells expressing GFP-ORP3 were mock treated with DMSO or with the pan-PKC inhibitor Gö6983 (20µM) for 2 hr. Both groups of cells were then stimulated with thapsigargin (1µM) and imaged live. Images show a representative cell after 5 min thapsigargin treatment. Bar = 10µm. (B) Percentage of ORP3 engaged with the PM from control and Gö6983 treated cells after thapsigargin treatment. Data were collected from 5 cells per group. (C) Quantification of focal adhesion size in control and Gö6983 treated cells, fixed and stained for vinculin as a marker for FA, N for control = 330 and for Gö6983 = 372 FA. Data were collected from 5 cells per group ( D) Cells co-expressing mCherry-IQSec1 and GFP-ORP3 were incubated with PMA in the presence or absence of Gö6983. ORP3 immunoprecipitates were then probed for IQSec1. This experiment was done twice and a similar trend was noted. (E) Lysates of cells expressing mCherry-ORP3 were incubated (or not) with λ-phosphatase to dephosphorylate ORP3, then precipitated with GST alone, or a GST fusion containing IQSec1 residues 152-212. This experiment was done twice and a similar trend was noted. (F) Arf5 activity was measured by pulldown in the presence and absence of the PKC inhibitor Gö6983. (G) Quantitation of Arf5 activity, corresponding to panel ‘F’. Data were compiled from three independent experiments. (H) Quantification cell migration out of spheroids from parental MDA-MB-231 cells or cells stably expressing-ORP3-FL. One batch of spheroids from each group was left untreated, and a second batch was treated with Gö6983 embedded in 3D collagen and imaged live for 22hrs. Data was collected from 7 spheroids from both groups

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: Phosphoregulation of ORP3 recruitment to the PM (A) MDA-MB-231cells expressing GFP-ORP3 were mock treated with DMSO or with the pan-PKC inhibitor Gö6983 (20µM) for 2 hr. Both groups of cells were then stimulated with thapsigargin (1µM) and imaged live. Images show a representative cell after 5 min thapsigargin treatment. Bar = 10µm. (B) Percentage of ORP3 engaged with the PM from control and Gö6983 treated cells after thapsigargin treatment. Data were collected from 5 cells per group. (C) Quantification of focal adhesion size in control and Gö6983 treated cells, fixed and stained for vinculin as a marker for FA, N for control = 330 and for Gö6983 = 372 FA. Data were collected from 5 cells per group ( D) Cells co-expressing mCherry-IQSec1 and GFP-ORP3 were incubated with PMA in the presence or absence of Gö6983. ORP3 immunoprecipitates were then probed for IQSec1. This experiment was done twice and a similar trend was noted. (E) Lysates of cells expressing mCherry-ORP3 were incubated (or not) with λ-phosphatase to dephosphorylate ORP3, then precipitated with GST alone, or a GST fusion containing IQSec1 residues 152-212. This experiment was done twice and a similar trend was noted. (F) Arf5 activity was measured by pulldown in the presence and absence of the PKC inhibitor Gö6983. (G) Quantitation of Arf5 activity, corresponding to panel ‘F’. Data were compiled from three independent experiments. (H) Quantification cell migration out of spheroids from parental MDA-MB-231 cells or cells stably expressing-ORP3-FL. One batch of spheroids from each group was left untreated, and a second batch was treated with Gö6983 embedded in 3D collagen and imaged live for 22hrs. Data was collected from 7 spheroids from both groups

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Multiple Displacement Amplification, Expressing, Staining, Marker, Incubation, Activity Assay, Quantitation Assay, Migration, Stable Transfection

    IQSec1 activates ARF5 to promote FA disassembly (A) Control and ARF5 depleted cells stained for β1-integrin (green) and FAK (red). Bar = 10µm. (B) FA disassembly rates in cells expressing GFP-Paxillin. Cells depleted of IQSec1 were transfected with plasmids expressing rapid cycling (RC) mutants of ARF6, ARF5 or ARF1. N for control = 520, IQSec1 KD = 314, IQSec1 KD and ARF5 RC = 570, IQSec1 KD and Arf6 RC = 252, IQSec1 KD and ARF1 RC = 362 focal adhesions. Data were collected from 5 cells per group. (C) Invasion of control vs. ARF5-depleted cells through a Matrigel plug. Data were compiled from 3 independent experiments. (D and E) Pulldown assay for ARF5 activity. HEK293 cells expressing ARF5 and mCherry-IQSec1 or an mCherry-IQSec1E606K mutant were cultured in serum and calcium free media for 3 hrs, then stimulated with thapsigargin [T] (1µM) in the presence of calcium (1mM) for 30 minutes. Cells lysates were then incubated with GST-GGA3 beads to precipitate active, GTP-bound ARF5. (D) A representative blot showing enhanced activation of ARF5 by thapsigargin in the presence of IQSec1 WT, but not catalytically inactive IQSec1 E606K mutant (E) Quantitation of results from three independent experiments shown in ‘D’. (F) Representative blot showing decreased ARF5 activity in cells treated with the Orai1 inhibitor BTP2 (25µM). (G) Quantitation of results from three independent experiments shown in ‘F’. (H) Quantification of FA size from control and STIM1 depleted cells, fixed and stained for vinculin as a marker for FAs, N for control = 330 and for STIM1 KD= 538 FA. 10 cells were analyzed per group. (I) Quantification cell migration out of spheroids derived from control and STIM1 depleted cells. Data was collected from 8 spheroids from each group.

    Journal: bioRxiv

    Article Title: Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

    doi: 10.1101/866392

    Figure Lengend Snippet: IQSec1 activates ARF5 to promote FA disassembly (A) Control and ARF5 depleted cells stained for β1-integrin (green) and FAK (red). Bar = 10µm. (B) FA disassembly rates in cells expressing GFP-Paxillin. Cells depleted of IQSec1 were transfected with plasmids expressing rapid cycling (RC) mutants of ARF6, ARF5 or ARF1. N for control = 520, IQSec1 KD = 314, IQSec1 KD and ARF5 RC = 570, IQSec1 KD and Arf6 RC = 252, IQSec1 KD and ARF1 RC = 362 focal adhesions. Data were collected from 5 cells per group. (C) Invasion of control vs. ARF5-depleted cells through a Matrigel plug. Data were compiled from 3 independent experiments. (D and E) Pulldown assay for ARF5 activity. HEK293 cells expressing ARF5 and mCherry-IQSec1 or an mCherry-IQSec1E606K mutant were cultured in serum and calcium free media for 3 hrs, then stimulated with thapsigargin [T] (1µM) in the presence of calcium (1mM) for 30 minutes. Cells lysates were then incubated with GST-GGA3 beads to precipitate active, GTP-bound ARF5. (D) A representative blot showing enhanced activation of ARF5 by thapsigargin in the presence of IQSec1 WT, but not catalytically inactive IQSec1 E606K mutant (E) Quantitation of results from three independent experiments shown in ‘D’. (F) Representative blot showing decreased ARF5 activity in cells treated with the Orai1 inhibitor BTP2 (25µM). (G) Quantitation of results from three independent experiments shown in ‘F’. (H) Quantification of FA size from control and STIM1 depleted cells, fixed and stained for vinculin as a marker for FAs, N for control = 330 and for STIM1 KD= 538 FA. 10 cells were analyzed per group. (I) Quantification cell migration out of spheroids derived from control and STIM1 depleted cells. Data was collected from 8 spheroids from each group.

    Article Snippet: Chemicals and Inhibitors Thapsigargin, Phorbol 12-myristate 13-acetate (PMA) and YM 58483 (BTP2) were purchased from Tocris (Bristol, UK).

    Techniques: Staining, Expressing, Transfection, Activity Assay, Mutagenesis, Cell Culture, Incubation, Activation Assay, Quantitation Assay, Marker, Migration, Derivative Assay

    Hyperthermia increases ATP-tumor killing activity by enhancing P2X7 pore formation independently of Ca 2+ influx and pannexin/connexin interaction (A) P2X7 functionality upon ATP/hyperthermia treatment measured by etidium bromide (EtBr) uptake. Cells were left untreated or treated with ATP (1 mM) for 15 min at 37°C or 40°C, followed by whole cell fluorescence measurement (AUF), as described in Material and Methods. (B) Cells were pre-incubated with BAPTA-AM, thapsigargin (TG) or Carbenoxolone (CBX) prior to heat-ATP pulse treatment, followed by western blot analysis of AKT/PRAS40/mTOR signaling pathway. Cells treated with media served as control. (C) NC (negative control) and P2X7 KD (P2X7-deficient) cells were exposed to ATP for 15 min at 37°C or 40°C and extracellular and intracellular adenine nucleotide levels were determined by HPLC. *p

    Journal: Oncotarget

    Article Title: Hyperthermia and associated changes in membrane fluidity potentiate P2X7 activation to promote tumor cell death

    doi: 10.18632/oncotarget.18595

    Figure Lengend Snippet: Hyperthermia increases ATP-tumor killing activity by enhancing P2X7 pore formation independently of Ca 2+ influx and pannexin/connexin interaction (A) P2X7 functionality upon ATP/hyperthermia treatment measured by etidium bromide (EtBr) uptake. Cells were left untreated or treated with ATP (1 mM) for 15 min at 37°C or 40°C, followed by whole cell fluorescence measurement (AUF), as described in Material and Methods. (B) Cells were pre-incubated with BAPTA-AM, thapsigargin (TG) or Carbenoxolone (CBX) prior to heat-ATP pulse treatment, followed by western blot analysis of AKT/PRAS40/mTOR signaling pathway. Cells treated with media served as control. (C) NC (negative control) and P2X7 KD (P2X7-deficient) cells were exposed to ATP for 15 min at 37°C or 40°C and extracellular and intracellular adenine nucleotide levels were determined by HPLC. *p

    Article Snippet: Reagents and antibodies Carbenoxolone (CBX), BAPTA-AM and thapsigargin (TG) were purchased from Tocris Bioscience (Ellisville, MO).

    Techniques: Activity Assay, Fluorescence, Incubation, Western Blot, Negative Control, High Performance Liquid Chromatography

    GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with thapsigargin abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Distinct roles of ?- and ?CaMKII in controlling long-term potentiation of GABAA-receptor mediated transmission in murine Purkinje cells

    doi: 10.3389/fncel.2014.00016

    Figure Lengend Snippet: GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with thapsigargin abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.

    Article Snippet: PHARMACOLOGY Baclofen (2 μM), cyclosporin A (5 μM), KN-93 (2 μM), SCH50911 (10 μM), KT 5720 (0.2 μM), and thapsigargin (10 μM) were obtained from Tocris Biosciences (Bristol, UK).

    Techniques: Inhibition, Activation Assay, Knock-Out, Mouse Assay

    Astrocytic SOCE is mediated by Orai channels in combination with Stim1. (a) Schematic illustration of astrocyte Ca 2+ imaging in inverted microscope using ratiometric Ca 2+ dye, Fura-2 AM. (b) Experimental protocol of Ca 2+ imaging. Ca 2+ free HEPES solution containing 200 nM EGTA was bath applied for 1200 s. Meanwhile, 1 µM thapsigargin, the SERCA inhibitor, was applied for 120 s after 60 s baseline to induce Ca 2+ release from ER. External solution was changed to the normal HEPES solution containing 2 mM Ca 2+ at 1200 s. (c) Left top, Fura-2 AM loaded cells. Left bottom, Orai1, Orai2, and Orai3 shRNA co-transfected cells expressing mCherry or EGFP. Right top, representative Ca 2+ imaging 340/380 ratio images taken at 1 min, 3 min, 20 min, 25 min. Right bottom, representative 340/380 ratio traces of two cells. Orai1/2/3 shRNA transfected cell is indicated with red mark and endogenous control was indicated with yellow mark. (d~j) Average of total Ca 2+ imaging traces and scatter plots of ER Ca 2+ release (Release), store operated Ca 2+ entry (Entry) and Entry/Release ratio (Ratio) of primary cultured astrocytes transfected with scrambled shRNA (d), Orai1 shRNA (e), Orai2 shRNA (f), Orai3 shRNA (g), TrpC1 shRNA (h), Orai1/2/3 shRNA (i), Stim1 shRNA (j). Scale bar indicates 0.5 for y axis and 100s for x axis. Tg: thapsigargin.

    Journal: Experimental Neurobiology

    Article Title: Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes

    doi: 10.5607/en.2017.26.1.42

    Figure Lengend Snippet: Astrocytic SOCE is mediated by Orai channels in combination with Stim1. (a) Schematic illustration of astrocyte Ca 2+ imaging in inverted microscope using ratiometric Ca 2+ dye, Fura-2 AM. (b) Experimental protocol of Ca 2+ imaging. Ca 2+ free HEPES solution containing 200 nM EGTA was bath applied for 1200 s. Meanwhile, 1 µM thapsigargin, the SERCA inhibitor, was applied for 120 s after 60 s baseline to induce Ca 2+ release from ER. External solution was changed to the normal HEPES solution containing 2 mM Ca 2+ at 1200 s. (c) Left top, Fura-2 AM loaded cells. Left bottom, Orai1, Orai2, and Orai3 shRNA co-transfected cells expressing mCherry or EGFP. Right top, representative Ca 2+ imaging 340/380 ratio images taken at 1 min, 3 min, 20 min, 25 min. Right bottom, representative 340/380 ratio traces of two cells. Orai1/2/3 shRNA transfected cell is indicated with red mark and endogenous control was indicated with yellow mark. (d~j) Average of total Ca 2+ imaging traces and scatter plots of ER Ca 2+ release (Release), store operated Ca 2+ entry (Entry) and Entry/Release ratio (Ratio) of primary cultured astrocytes transfected with scrambled shRNA (d), Orai1 shRNA (e), Orai2 shRNA (f), Orai3 shRNA (g), TrpC1 shRNA (h), Orai1/2/3 shRNA (i), Stim1 shRNA (j). Scale bar indicates 0.5 for y axis and 100s for x axis. Tg: thapsigargin.

    Article Snippet: Thapsigargin (Tocris) was used as a sarcoendoplasmic reticulum calcium transport ATPase (SERCA) inhibitor.

    Techniques: Imaging, Inverted Microscopy, shRNA, Transfection, Expressing, Cell Culture