thapsigargin  (Alomone Labs)


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    Name:
    Thapsigargin
    Description:
    Thapsigargin is an extremely tight binding inhibitor of intracellular Ca2 pumps but initially described as a tumor promoting agent which induces rapid Ca2 release from intracellular stores In addition the thapsigargin induced depletion of Ca2 stores causes apoptosis in most cell lines
    Catalog Number:
    T-650
    Price:
    63.0
    Category:
    Small Molecule
    Source:
    Thapsia garganica.
    Applications:
    0
    Purity:
    >99% (HPLC)
    Size:
    0 65 mg
    Format:
    Lyophilized/solid.
    Formula:
    C34H50O12
    Molecular Weight:
    650.7
    Molecule Name:
    (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-oxobutox y)azuleno[4,5-b]furan-7-yl octanoate.
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    Structured Review

    Alomone Labs thapsigargin
    Thapsigargin
    Thapsigargin is an extremely tight binding inhibitor of intracellular Ca2 pumps but initially described as a tumor promoting agent which induces rapid Ca2 release from intracellular stores In addition the thapsigargin induced depletion of Ca2 stores causes apoptosis in most cell lines
    https://www.bioz.com/result/thapsigargin/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    thapsigargin - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Staphylococcal leukotoxins trigger free intracellular Ca2+ rise in neurones, signalling through acidic stores and activation of store-operated channels"

    Article Title: Staphylococcal leukotoxins trigger free intracellular Ca2+ rise in neurones, signalling through acidic stores and activation of store-operated channels

    Journal: Cellular Microbiology

    doi: 10.1111/cmi.12069

    Both extracellular and intracellular Ca 2+ stores contribute to leukotoxin-induced [Ca 2+ ] i changes in cerebellar neurones. A. Averaged traces of four recordings obtained at different extracellular Ca 2+ concentrations ([Ca 2+ ] e ) showing that, in a Ca 2+ -free medium, neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca 2+ ] e (3 μM), an increase in free [Ca 2+ ] i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 μM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 μM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 μM thapsigargin; two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 μM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. C. Lysosomal destruction by 0.2 μM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca 2+ ] i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke.
    Figure Legend Snippet: Both extracellular and intracellular Ca 2+ stores contribute to leukotoxin-induced [Ca 2+ ] i changes in cerebellar neurones. A. Averaged traces of four recordings obtained at different extracellular Ca 2+ concentrations ([Ca 2+ ] e ) showing that, in a Ca 2+ -free medium, neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca 2+ ] e (3 μM), an increase in free [Ca 2+ ] i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 μM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 μM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 μM thapsigargin; two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 μM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. C. Lysosomal destruction by 0.2 μM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca 2+ ] i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke.

    Techniques Used:

    2) Product Images from "Kisspeptin Activation of TRPC4 Channels in Female GnRH Neurons Requires PIP2 Depletion and cSrc Kinase Activation"

    Article Title: Kisspeptin Activation of TRPC4 Channels in Female GnRH Neurons Requires PIP2 Depletion and cSrc Kinase Activation

    Journal: Endocrinology

    doi: 10.1210/en.2013-1180

    Ca 2+ store depletion does not affect TRPC channel activation by kisspeptin. A–C, Representative recordings showing the kisspeptin (Kp-10)-induced inward current after a GnRH neuron had been exposed to thapsigargin (Tg, 1μM) for 10 minutes
    Figure Legend Snippet: Ca 2+ store depletion does not affect TRPC channel activation by kisspeptin. A–C, Representative recordings showing the kisspeptin (Kp-10)-induced inward current after a GnRH neuron had been exposed to thapsigargin (Tg, 1μM) for 10 minutes

    Techniques Used: Activation Assay

    3) Product Images from "Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters 1"

    Article Title: Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.112.100255

    Germ cells are viable in the SST and display spontaneous Ca 2+ oscillations. Ca 2+ recordings from germ cells in the SST corresponding to the addition of 10 μM thapsigargin ( A ) or 120 mM KCl ( B ). C ) Fluorescence traces obtained from the four cells
    Figure Legend Snippet: Germ cells are viable in the SST and display spontaneous Ca 2+ oscillations. Ca 2+ recordings from germ cells in the SST corresponding to the addition of 10 μM thapsigargin ( A ) or 120 mM KCl ( B ). C ) Fluorescence traces obtained from the four cells

    Techniques Used: Fluorescence

    4) Product Images from "Calcium-Activated Sustained Firing Responses Distinguish Accessory from Main Olfactory Bulb Mitral Cells"

    Article Title: Calcium-Activated Sustained Firing Responses Distinguish Accessory from Main Olfactory Bulb Mitral Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4397-11.2012

    Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p
    Figure Legend Snippet: Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p

    Techniques Used: Mass Spectrometry, Incubation, MANN-WHITNEY

    5) Product Images from "Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals"

    Article Title: Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals

    Journal: Neuron

    doi: 10.1016/j.neuron.2017.01.010

    SERCA function is necessary for activity-driven ER Ca 2+ uptake (A) Representative presynaptic responses of ER-GCaMP6-150 to 20AP (20Hz) or (B) ER-GCaMP6-210 to a single AP stimulus before and after 5 min of CPA (50 µM) application (black and red, respectively). Average of single AP Δ[Ca 2+ ] ER responses before CPA was 5.9 ± 1.3 µM (n=5), which was reduced to 0.2 ± 0.2 µM after CPA treatment (n=3). (C) Box plots showing average and single-cell calibrated peak responses of neurons stimulated with 20AP (20Hz) before and after 5 min of treatment with CPA (n=10), thapsigargin (TG, 1 µM, n=9) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 50 µM, n=9).
    Figure Legend Snippet: SERCA function is necessary for activity-driven ER Ca 2+ uptake (A) Representative presynaptic responses of ER-GCaMP6-150 to 20AP (20Hz) or (B) ER-GCaMP6-210 to a single AP stimulus before and after 5 min of CPA (50 µM) application (black and red, respectively). Average of single AP Δ[Ca 2+ ] ER responses before CPA was 5.9 ± 1.3 µM (n=5), which was reduced to 0.2 ± 0.2 µM after CPA treatment (n=3). (C) Box plots showing average and single-cell calibrated peak responses of neurons stimulated with 20AP (20Hz) before and after 5 min of treatment with CPA (n=10), thapsigargin (TG, 1 µM, n=9) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 50 µM, n=9).

    Techniques Used: Activity Assay

    6) Product Images from "Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones"

    Article Title: Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones

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

    doi: 10.1523/JNEUROSCI.0550-11.2011

    Regulation of CamKII activation and translocation by IP3Rs. (a) FRET responses in the presence of 0.1% DMSO (n=8), 20 μM thapsigargin in DMSO (Tg, n=5) 0.1 μM xestospongin C in DMSO (Xesto, n=9), or with neuronal expression of IP3R RNAi
    Figure Legend Snippet: Regulation of CamKII activation and translocation by IP3Rs. (a) FRET responses in the presence of 0.1% DMSO (n=8), 20 μM thapsigargin in DMSO (Tg, n=5) 0.1 μM xestospongin C in DMSO (Xesto, n=9), or with neuronal expression of IP3R RNAi

    Techniques Used: Activation Assay, Translocation Assay, Expressing

    7) Product Images from "A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells"

    Article Title: A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.581678

    Increased SOCE correlates with increased cell proliferation in human colon carcinoma cells. A, SOCE in normal colon cancer cell lines. SOCE was recorded by Ca 2+ imaging of fura-2-loaded cells treated with thapsigargin (1 μ m , 10 min) in Ca 2+ -free
    Figure Legend Snippet: Increased SOCE correlates with increased cell proliferation in human colon carcinoma cells. A, SOCE in normal colon cancer cell lines. SOCE was recorded by Ca 2+ imaging of fura-2-loaded cells treated with thapsigargin (1 μ m , 10 min) in Ca 2+ -free

    Techniques Used: Imaging

    Effects of La 3+ on I SOC in normal (NCM460) and colon carcinoma (HT29) cells. I SOC were activated by passive Ca 2+ store depletion with extracellular thapsigargin (1 μ m ). For NCM460 cells, representative I-V relationships of I SOC , in the absence
    Figure Legend Snippet: Effects of La 3+ on I SOC in normal (NCM460) and colon carcinoma (HT29) cells. I SOC were activated by passive Ca 2+ store depletion with extracellular thapsigargin (1 μ m ). For NCM460 cells, representative I-V relationships of I SOC , in the absence

    Techniques Used:

    SOCs activated by thapsigargin and ATP in normal (NCM460) and colon carcinoma (HT29) cells. A, average time course plot of I CRAC activated with extracellular thapsigargin (1 μ m ) in normal colon cells ( n = 17). B, average time course plots of
    Figure Legend Snippet: SOCs activated by thapsigargin and ATP in normal (NCM460) and colon carcinoma (HT29) cells. A, average time course plot of I CRAC activated with extracellular thapsigargin (1 μ m ) in normal colon cells ( n = 17). B, average time course plots of

    Techniques Used:

    8) Product Images from "The Effects of Diuretics on Intracellular Ca2+ Dynamics of Arteriole Smooth Muscles as Revealed by Laser Confocal Microscopy"

    Article Title: The Effects of Diuretics on Intracellular Ca2+ Dynamics of Arteriole Smooth Muscles as Revealed by Laser Confocal Microscopy

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.09006

    Time courses for the spironolactone-induced [Ca 2+ ] i dynamics under various modulations. Temporal changes of [Ca 2+ ] i for three ROIs are depicted (black, gray and dotted lines). ( A ) positive controls (only spironolactone stimulation). The initial acute increase (long arrow) and a gradual decline (small thick arrow) are shown. ( B ) spironolactone-induced [Ca 2+ ] i dynamics under extracellular Ca 2+ -free conditions ([Ca 2+ ] o -free). ( C ) spironolactone-induced [Ca 2+ ] i dynamics in the presence of Gd 3+ (100 µM). ( D ) spironolactone-induced [Ca 2+ ] i dynamics after depleting intracellular Ca 2+ stores by treatment with thapsigargin (2 µM). A gradual decline (small thick arrow) are shown.
    Figure Legend Snippet: Time courses for the spironolactone-induced [Ca 2+ ] i dynamics under various modulations. Temporal changes of [Ca 2+ ] i for three ROIs are depicted (black, gray and dotted lines). ( A ) positive controls (only spironolactone stimulation). The initial acute increase (long arrow) and a gradual decline (small thick arrow) are shown. ( B ) spironolactone-induced [Ca 2+ ] i dynamics under extracellular Ca 2+ -free conditions ([Ca 2+ ] o -free). ( C ) spironolactone-induced [Ca 2+ ] i dynamics in the presence of Gd 3+ (100 µM). ( D ) spironolactone-induced [Ca 2+ ] i dynamics after depleting intracellular Ca 2+ stores by treatment with thapsigargin (2 µM). A gradual decline (small thick arrow) are shown.

    Techniques Used:

    9) Product Images from "CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca2+ entry-stimulated respiration"

    Article Title: CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca2+ entry-stimulated respiration

    Journal: Scientific Reports

    doi: 10.1038/srep42993

    Mitochondrial Ca 2+ uptake during SOCE and SOCE-stimulation of respiration is reduced in GDAP1-KD cells. ( A ) Analysis of MCU levels by Western blot. Protein extracts were obtained 72 hours after transfection of N2a cells with either shScr or shMcu. Primary antibodies used were α-MCU and α-βATPase as a control. MCU protein levels drop to 56, 2 ± 8, 3% of control values. ( B ) Fura-2 [Ca 2+ ] i signals and 4mt-D3cpv mitochondrial calcium signals in N2a cells transfected with shScr or shMcu upon addition of 25 μM ATP where indicated. ( C ) Lyn-D3cpv subplasmalemmal Ca 2+ signals were measured in N2a cells transfected with shScr or shMcu upon addition of 2 mM Ca 2+ in Ca 2+ -free medium with 5 μM Thapsigargin (Tg). Data were obtained from 3 independent experiments (n = 9–16 cells). ( D) Quantification of SOCE amplitude as ΔRatio (F510/F440) ± SEM for each condition. ( E) SOCE response in control pLKO and GDAP1 -KD neuroblastoma cells, in presence or absence of DNP (0.25 mM). Fura-2 [Ca 2+ ] i signals were measured upon addition of 5 μM Tg in Ca 2+ -free medium and 2 mM CaCl 2 where indicated. DNP was added 2 min before Ca 2+ addition. Traces were obtained averaging at least 250 cells from at least 4 independent experiments. ( F) Quantification of SOCE amplitude as ΔRatio (F340/F380) ± SEM for each cell line and condition. ( G) Oxygen consumption rate expressed as percentage of basal OCR in control pLKO and GDAP1 -KD cells, showing the sequential injection of carbachol (Cch, 50 μM), Ca 2+ (2 mM) and metabolic inhibitors. ( H , I) Quantification of % OCR 6 min after carbachol addition and 3 min after calcium addition respectively. Data were obtained from at least 8 independent experiments (n = 27–50). All data are normalized to the initial values and are expressed as mean ± SEM. Means were compared using one-way or two-way ANOVA, *p
    Figure Legend Snippet: Mitochondrial Ca 2+ uptake during SOCE and SOCE-stimulation of respiration is reduced in GDAP1-KD cells. ( A ) Analysis of MCU levels by Western blot. Protein extracts were obtained 72 hours after transfection of N2a cells with either shScr or shMcu. Primary antibodies used were α-MCU and α-βATPase as a control. MCU protein levels drop to 56, 2 ± 8, 3% of control values. ( B ) Fura-2 [Ca 2+ ] i signals and 4mt-D3cpv mitochondrial calcium signals in N2a cells transfected with shScr or shMcu upon addition of 25 μM ATP where indicated. ( C ) Lyn-D3cpv subplasmalemmal Ca 2+ signals were measured in N2a cells transfected with shScr or shMcu upon addition of 2 mM Ca 2+ in Ca 2+ -free medium with 5 μM Thapsigargin (Tg). Data were obtained from 3 independent experiments (n = 9–16 cells). ( D) Quantification of SOCE amplitude as ΔRatio (F510/F440) ± SEM for each condition. ( E) SOCE response in control pLKO and GDAP1 -KD neuroblastoma cells, in presence or absence of DNP (0.25 mM). Fura-2 [Ca 2+ ] i signals were measured upon addition of 5 μM Tg in Ca 2+ -free medium and 2 mM CaCl 2 where indicated. DNP was added 2 min before Ca 2+ addition. Traces were obtained averaging at least 250 cells from at least 4 independent experiments. ( F) Quantification of SOCE amplitude as ΔRatio (F340/F380) ± SEM for each cell line and condition. ( G) Oxygen consumption rate expressed as percentage of basal OCR in control pLKO and GDAP1 -KD cells, showing the sequential injection of carbachol (Cch, 50 μM), Ca 2+ (2 mM) and metabolic inhibitors. ( H , I) Quantification of % OCR 6 min after carbachol addition and 3 min after calcium addition respectively. Data were obtained from at least 8 independent experiments (n = 27–50). All data are normalized to the initial values and are expressed as mean ± SEM. Means were compared using one-way or two-way ANOVA, *p

    Techniques Used: Western Blot, Transfection, Injection

    10) Product Images from "Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones"

    Article Title: Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones

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

    doi: 10.1523/JNEUROSCI.0550-11.2011

    Regulation of CamKII activation and translocation by IP3Rs. (a) FRET responses in the presence of 0.1% DMSO (n=8), 20 μM thapsigargin in DMSO (Tg, n=5) 0.1 μM xestospongin C in DMSO (Xesto, n=9), or with neuronal expression of IP3R RNAi
    Figure Legend Snippet: Regulation of CamKII activation and translocation by IP3Rs. (a) FRET responses in the presence of 0.1% DMSO (n=8), 20 μM thapsigargin in DMSO (Tg, n=5) 0.1 μM xestospongin C in DMSO (Xesto, n=9), or with neuronal expression of IP3R RNAi

    Techniques Used: Activation Assay, Translocation Assay, Expressing

    11) Product Images from "Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle, et al. Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle"

    Article Title: Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle, et al. Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

    Journal: Aging Cell

    doi: 10.1111/acel.13134

    Role of luminal SR calcium on T‐type Ca V 3.2‐RyR axis. Effects of different concentrations of thapsigargin on Ca 2+ spark frequency (a, left) and fraction of cells producing Ca 2+ sparks (a, right) in Ca v 1.2 +/+ VSMCs from young mice. Effects of different concentrations of thapsigargin on Ca 2+ spark frequency (b, left) and fraction of cells producing Ca 2+ sparks (b, right) in VSMCs from Ca v 1.2 −/− (SMAKO) mice. (c), overlay of the data for Ca 2+ spark frequency (left) and fraction of cells producing Ca 2+ sparks (right). Cells were isolated from 4 mice in each group; 30–35 cells were recorded and analyzed from each mouse. (d), time course of Ca 2+ fluorescence changes in the cellular ROI in a wild‐type (Ca V 1.2 +/+ ) Fluo‐4‐AM–loaded VSMC induced by 10 mM caffeine (upper panel) and Ca 2+ fluorescence plots (lower panel). (e), the same as (d), but in Ca V 1.2 −/− VSMC. (f), summary of the 10 mM caffeine‐induced Ca 2+ peaks in wild‐type versus Ca V 1.2 −/− VSMCs. n = 7 cells from 3 mice, 2–3 cells were recorded and analyzed from each mouse. *, p
    Figure Legend Snippet: Role of luminal SR calcium on T‐type Ca V 3.2‐RyR axis. Effects of different concentrations of thapsigargin on Ca 2+ spark frequency (a, left) and fraction of cells producing Ca 2+ sparks (a, right) in Ca v 1.2 +/+ VSMCs from young mice. Effects of different concentrations of thapsigargin on Ca 2+ spark frequency (b, left) and fraction of cells producing Ca 2+ sparks (b, right) in VSMCs from Ca v 1.2 −/− (SMAKO) mice. (c), overlay of the data for Ca 2+ spark frequency (left) and fraction of cells producing Ca 2+ sparks (right). Cells were isolated from 4 mice in each group; 30–35 cells were recorded and analyzed from each mouse. (d), time course of Ca 2+ fluorescence changes in the cellular ROI in a wild‐type (Ca V 1.2 +/+ ) Fluo‐4‐AM–loaded VSMC induced by 10 mM caffeine (upper panel) and Ca 2+ fluorescence plots (lower panel). (e), the same as (d), but in Ca V 1.2 −/− VSMC. (f), summary of the 10 mM caffeine‐induced Ca 2+ peaks in wild‐type versus Ca V 1.2 −/− VSMCs. n = 7 cells from 3 mice, 2–3 cells were recorded and analyzed from each mouse. *, p

    Techniques Used: Mouse Assay, Isolation, Fluorescence

    12) Product Images from "Homer regulates calcium signalling in growth cone turning"

    Article Title: Homer regulates calcium signalling in growth cone turning

    Journal: Neural Development

    doi: 10.1186/1749-8104-4-29

    Homer1 knockdown perturbs dorsal root ganglia growth cone calcium dynamics . (A) Control (closed red circles) morphant growth cones (n = 9) exposed to a brain derived neurotrophic factor (BDNF) micro-gradient show significantly more calcium flux than Homer1 (closed green circles) morphant growth cones (n = 16). (B) Bath application of thapsigargin 2 minutes after establishment of a BDNF micro-gradient elicited a robust rise in intracellular calcium in Homer morphants (hom morph) but failed to elicit any rise in calcium in control morphants (ctrl morph). Error bars indicate standard error of the mean.
    Figure Legend Snippet: Homer1 knockdown perturbs dorsal root ganglia growth cone calcium dynamics . (A) Control (closed red circles) morphant growth cones (n = 9) exposed to a brain derived neurotrophic factor (BDNF) micro-gradient show significantly more calcium flux than Homer1 (closed green circles) morphant growth cones (n = 16). (B) Bath application of thapsigargin 2 minutes after establishment of a BDNF micro-gradient elicited a robust rise in intracellular calcium in Homer morphants (hom morph) but failed to elicit any rise in calcium in control morphants (ctrl morph). Error bars indicate standard error of the mean.

    Techniques Used: Derivative Assay

    13) Product Images from "Ca2+ store dynamics determines the pattern of activation of the store-operated Ca2+ current ICRAC in response to InsP3 in rat basophilic leukaemia cells"

    Article Title: Ca2+ store dynamics determines the pattern of activation of the store-operated Ca2+ current ICRAC in response to InsP3 in rat basophilic leukaemia cells

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.2000.t01-2-00283.x

    Graded I CRAC can be obtained in the presence of moderate Ca 2+ buffer because of SERCA uptake and inhibition of the Ins P 3 receptor A shows recordings for a cell dialysed with 30 μM Ins P 3 -F and 0.6 mM EGTA (•) and for a cell dialysed with this solution but supplemented with 2 μM thapsigargin (thap), a SERCA blocker. Note the dramatic increase in the size of I CRAC following inhibition of the pumps. B , summary of amplitudes, delays and times to peak for experiments as in A . C , cells dialysed with Ins P 3 and 1 mg ml −1 heparin (an inhibitor of the Ins P 3 receptor) can produce a wide range of different I CRAC amplitudes, in spite of the presence of a maximal Ins P 3 concentration. This experiment will depend on the relative speed of entry of Ins P 3 and heparin into the cytosol (proportional to series resistance), subsequent diffusion in the cytosol and access to the Ins P 3 receptors. Cells were dialysed with a solution in which Ca 2+ was buffered at 225 nM (3 mM Ca-EGTA and 2 mM EGTA). The decay in the current reflects store refilling because it could be reduced by inclusion of thapsigargin in the pipette (5 cells; data not shown). Dialysis with heparin fails to alter the activation of I CRAC ), indicating that it is not interfering directly with the activation mechanism itself nor with the CRAC channels.
    Figure Legend Snippet: Graded I CRAC can be obtained in the presence of moderate Ca 2+ buffer because of SERCA uptake and inhibition of the Ins P 3 receptor A shows recordings for a cell dialysed with 30 μM Ins P 3 -F and 0.6 mM EGTA (•) and for a cell dialysed with this solution but supplemented with 2 μM thapsigargin (thap), a SERCA blocker. Note the dramatic increase in the size of I CRAC following inhibition of the pumps. B , summary of amplitudes, delays and times to peak for experiments as in A . C , cells dialysed with Ins P 3 and 1 mg ml −1 heparin (an inhibitor of the Ins P 3 receptor) can produce a wide range of different I CRAC amplitudes, in spite of the presence of a maximal Ins P 3 concentration. This experiment will depend on the relative speed of entry of Ins P 3 and heparin into the cytosol (proportional to series resistance), subsequent diffusion in the cytosol and access to the Ins P 3 receptors. Cells were dialysed with a solution in which Ca 2+ was buffered at 225 nM (3 mM Ca-EGTA and 2 mM EGTA). The decay in the current reflects store refilling because it could be reduced by inclusion of thapsigargin in the pipette (5 cells; data not shown). Dialysis with heparin fails to alter the activation of I CRAC ), indicating that it is not interfering directly with the activation mechanism itself nor with the CRAC channels.

    Techniques Used: Inhibition, Concentration Assay, Diffusion-based Assay, Transferring, Activation Assay

    14) Product Images from "Prostaglandin E2 Inhibits Histamine-Evoked Ca2+"

    Article Title: Prostaglandin E2 Inhibits Histamine-Evoked Ca2+

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.117.109249

    Cyclic AMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 . (A) Effect of 8-Br-cAMP (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± S.E.M. from at least three experiments with one to three wells in each. (B) 8-Br-cAMP (10 mM, 20 minutes) had no effect on the Ca 2+ content of the intracellular stores as revealed by the increases in [Ca 2+ ] i evoked by addition of thapsigargin (1 μ M) or ionomycin (1 μ Μ) in Ca 2+ -free HBS. Results (percentages of responses without 8-Br-cAMP) are means ± S.E.M. from five experiments with three to four wells analyzed in each. (C) Effect of the indicated cyclic nucleotides (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by histamine (3 μ M). Results are means ± S.E.M. from three to five experiments with two to three wells in each. (D) Effect of NKH 477 (100 μ M, 5 minutes), forskolin (100 μ M, 5 minutes), 8-Br-cAMP (10 mM, 20 minutes), PGE 2 (10 μ Μ, 5 minutes), 8-Br-cGMP (10 mM, 20 minutes), R p-cAMPS (10 mM, 20 minutes), or 8-pCPT-2 ′ -O-Me-cAMP (10 mM, 20 minutes) alone or in combination on the peak Ca 2+ signals evoked by histamine (1 mM). Results (as percentages of the response to histamine alone) are means ± S.E.M. from three experiments with two to three wells in each. Results for R p-cAMPS are from a single experiment with three replicates, limited by the availability of this expensive analog. (E) Effects of the EPAC antagonists, ESI-09 and HJC0197 (10 μ M, 20 minutes), on the Ca 2+ signals evoked by histamine (3 μ M) added 5 minutes before and then during treatment with the indicated concentrations of PGE 2 . Results are expressed as percentages of the paired response to histamine alone (means ± S.E.M., n = 3–5; n = 2 for the antagonists with 1 and 3 nM PGE 2 , where error bars show ranges). (F) The results establish that cAMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 .
    Figure Legend Snippet: Cyclic AMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 . (A) Effect of 8-Br-cAMP (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± S.E.M. from at least three experiments with one to three wells in each. (B) 8-Br-cAMP (10 mM, 20 minutes) had no effect on the Ca 2+ content of the intracellular stores as revealed by the increases in [Ca 2+ ] i evoked by addition of thapsigargin (1 μ M) or ionomycin (1 μ Μ) in Ca 2+ -free HBS. Results (percentages of responses without 8-Br-cAMP) are means ± S.E.M. from five experiments with three to four wells analyzed in each. (C) Effect of the indicated cyclic nucleotides (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by histamine (3 μ M). Results are means ± S.E.M. from three to five experiments with two to three wells in each. (D) Effect of NKH 477 (100 μ M, 5 minutes), forskolin (100 μ M, 5 minutes), 8-Br-cAMP (10 mM, 20 minutes), PGE 2 (10 μ Μ, 5 minutes), 8-Br-cGMP (10 mM, 20 minutes), R p-cAMPS (10 mM, 20 minutes), or 8-pCPT-2 ′ -O-Me-cAMP (10 mM, 20 minutes) alone or in combination on the peak Ca 2+ signals evoked by histamine (1 mM). Results (as percentages of the response to histamine alone) are means ± S.E.M. from three experiments with two to three wells in each. Results for R p-cAMPS are from a single experiment with three replicates, limited by the availability of this expensive analog. (E) Effects of the EPAC antagonists, ESI-09 and HJC0197 (10 μ M, 20 minutes), on the Ca 2+ signals evoked by histamine (3 μ M) added 5 minutes before and then during treatment with the indicated concentrations of PGE 2 . Results are expressed as percentages of the paired response to histamine alone (means ± S.E.M., n = 3–5; n = 2 for the antagonists with 1 and 3 nM PGE 2 , where error bars show ranges). (F) The results establish that cAMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 .

    Techniques Used: Inhibition

    15) Product Images from "Calcium-Activated Sustained Firing Responses Distinguish Accessory from Main Olfactory Bulb Mitral Cells"

    Article Title: Calcium-Activated Sustained Firing Responses Distinguish Accessory from Main Olfactory Bulb Mitral Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4397-11.2012

    Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p
    Figure Legend Snippet: Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p

    Techniques Used: Incubation, MANN-WHITNEY

    16) Product Images from "Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes"

    Article Title: Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111275

    Carbachol evokes store-operated Ca 2+ entry in HEK-PR1 cells. (A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca 2+ . For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca 2+ -free HBS before restoration of extracellular Ca 2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca 2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca 2+ ([Ca 2+ ] e ) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.
    Figure Legend Snippet: Carbachol evokes store-operated Ca 2+ entry in HEK-PR1 cells. (A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca 2+ . For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca 2+ -free HBS before restoration of extracellular Ca 2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca 2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca 2+ ([Ca 2+ ] e ) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.

    Techniques Used: Incubation

    Lysosomes do not accumulate Ca 2+ entering cells via store-operated Ca 2+ entry evoked by carbachol. (A) Ca 2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP 3 Rs from which some Ca 2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca 2+ -free HBS alone (B) or with bafilomycin A 1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca 2+ signal attributable to Ca 2+ entry (ΔΔ[Ca 2+ ] i ) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca 2+ ] i evoked by CCh in normal or Ca 2+ -free HBS, with and without bafilomycin A 1 -treatment. Results (percentages of the responses to CCh alone in Ca 2+ -free HBS) are means ± S.E. from 4 experiments. * p
    Figure Legend Snippet: Lysosomes do not accumulate Ca 2+ entering cells via store-operated Ca 2+ entry evoked by carbachol. (A) Ca 2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP 3 Rs from which some Ca 2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca 2+ -free HBS alone (B) or with bafilomycin A 1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca 2+ signal attributable to Ca 2+ entry (ΔΔ[Ca 2+ ] i ) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca 2+ ] i evoked by CCh in normal or Ca 2+ -free HBS, with and without bafilomycin A 1 -treatment. Results (percentages of the responses to CCh alone in Ca 2+ -free HBS) are means ± S.E. from 4 experiments. * p

    Techniques Used:

    17) Product Images from "Store-operated calcium entry in vagal sensory nerves is independent of Orai channels"

    Article Title: Store-operated calcium entry in vagal sensory nerves is independent of Orai channels

    Journal: Brain research

    doi: 10.1016/j.brainres.2013.02.002

    Left , mean +/− SEM Ca 2+ responses of vagal neuron soma to thapsigargin (Thapsi, 10 μM, red, n=310), caffeine (Caff, 10mM, black, n=233) and vehicle (green, n=88) during changes in [Ca 2+ ] extracellular . Response to KCl (75mM) is included.
    Figure Legend Snippet: Left , mean +/− SEM Ca 2+ responses of vagal neuron soma to thapsigargin (Thapsi, 10 μM, red, n=310), caffeine (Caff, 10mM, black, n=233) and vehicle (green, n=88) during changes in [Ca 2+ ] extracellular . Response to KCl (75mM) is included.

    Techniques Used:

    (A) Left , mean +/− SEM Ca 2+ responses of vagal neurons that responded to the Ca 2+ addback following thapsigargin (1 μM, green, n=32 out of 108) or following thapsigargin (10 μM) in the absence (black, n=121 out of 310) or presence
    Figure Legend Snippet: (A) Left , mean +/− SEM Ca 2+ responses of vagal neurons that responded to the Ca 2+ addback following thapsigargin (1 μM, green, n=32 out of 108) or following thapsigargin (10 μM) in the absence (black, n=121 out of 310) or presence

    Techniques Used:

    Mean +/− SEM Ca 2+ responses of WT (n=111, red) and TRPA1−/ − (n=74, black) vagal neurons to thapsigargin (10μM). Blocked line denotes the 60-s application of agonist. Data are presented as mean change in 340/380 ratio as
    Figure Legend Snippet: Mean +/− SEM Ca 2+ responses of WT (n=111, red) and TRPA1−/ − (n=74, black) vagal neurons to thapsigargin (10μM). Blocked line denotes the 60-s application of agonist. Data are presented as mean change in 340/380 ratio as

    Techniques Used:

    Mean +/− SEM Ca 2+ responses of WT (n=7, red) and TRPA1−/ − (n=5, black) vagal neurites to thapsigargin (Thapsi, 10μM). Blocked line denotes the 60-s application of agonist. All neurite structures responded to KCl (75mM)
    Figure Legend Snippet: Mean +/− SEM Ca 2+ responses of WT (n=7, red) and TRPA1−/ − (n=5, black) vagal neurites to thapsigargin (Thapsi, 10μM). Blocked line denotes the 60-s application of agonist. All neurite structures responded to KCl (75mM)

    Techniques Used:

    18) Product Images from "Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum"

    Article Title: Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.116103

    Disruption of lysosomal Ca 2+ uptake with bafilomycin A 1 does not affect SOCE. ( A ) Transient increase in [Ca 2+ ] i evoked by thapsigargin (1 µM, solid bar) in HEK cells in nominally Ca 2+ -free HBS in control and bafilomycin A 1 -treated cells (Baf A 1 ; 1 µM, 1 h). ( B ) Summary results show effects of bafilomycin A 1 (1 µM, 1 h) on the peak increase in [Ca 2+ ] i evoked by thapsigargin (1 µM) in nominally Ca 2+ -free HBS. Results are means ± s.e.m. from three independent experiments. ( C ) Restoration of extracellular Ca 2+ (30 mM) to cells pre-treated with thapsigargin (1 µM, 15 min) in nominally Ca 2+ -free HBS to deplete intracellular Ca 2+ stores evokes SOCE. The response is indistinguishable in control cells and cells treated with bafilomycin A 1 (1 µM, 1 h). ( D ) SOCE after restoration of different concentrations of extracellular Ca 2+ ([Ca 2+ ] e ) to control and bafilomycin A 1 -treated cells (1 µM, 1 h). Results are means ± s.e.m. from three independent experiments. ( E ) TPEN (100 µM, 2 min) in nominally Ca 2+ -free HBS was used to reduce the free [Ca 2+ ] within the ER before restoration of the indicated concentrations of extracellular Ca 2+ to control or bafilomycin A 1 -treated cells (1 µM, 1 h). Results show the peak increase in [Ca 2+ ] i detected within 60 s after restoration of extracellular Ca 2+ . ( F ) Peak increase in [Ca 2+ ] i evoked by CCh (1 mM) in the presence of TPEN (100 µM, 2 min) with and without pre-incubation with bafilomycin A 1 . * P
    Figure Legend Snippet: Disruption of lysosomal Ca 2+ uptake with bafilomycin A 1 does not affect SOCE. ( A ) Transient increase in [Ca 2+ ] i evoked by thapsigargin (1 µM, solid bar) in HEK cells in nominally Ca 2+ -free HBS in control and bafilomycin A 1 -treated cells (Baf A 1 ; 1 µM, 1 h). ( B ) Summary results show effects of bafilomycin A 1 (1 µM, 1 h) on the peak increase in [Ca 2+ ] i evoked by thapsigargin (1 µM) in nominally Ca 2+ -free HBS. Results are means ± s.e.m. from three independent experiments. ( C ) Restoration of extracellular Ca 2+ (30 mM) to cells pre-treated with thapsigargin (1 µM, 15 min) in nominally Ca 2+ -free HBS to deplete intracellular Ca 2+ stores evokes SOCE. The response is indistinguishable in control cells and cells treated with bafilomycin A 1 (1 µM, 1 h). ( D ) SOCE after restoration of different concentrations of extracellular Ca 2+ ([Ca 2+ ] e ) to control and bafilomycin A 1 -treated cells (1 µM, 1 h). Results are means ± s.e.m. from three independent experiments. ( E ) TPEN (100 µM, 2 min) in nominally Ca 2+ -free HBS was used to reduce the free [Ca 2+ ] within the ER before restoration of the indicated concentrations of extracellular Ca 2+ to control or bafilomycin A 1 -treated cells (1 µM, 1 h). Results show the peak increase in [Ca 2+ ] i detected within 60 s after restoration of extracellular Ca 2+ . ( F ) Peak increase in [Ca 2+ ] i evoked by CCh (1 mM) in the presence of TPEN (100 µM, 2 min) with and without pre-incubation with bafilomycin A 1 . * P

    Techniques Used: Incubation

    19) Product Images from "Mitochondrial dysfunction induced by frataxin deficiency is associated with cellular senescence and abnormal calcium metabolism"

    Article Title: Mitochondrial dysfunction induced by frataxin deficiency is associated with cellular senescence and abnormal calcium metabolism

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00124

    FXN depletion results in increased endoplasmic reticulum (ER) stress. (A) Western blot analysis of ER stress marker BIP in control and FXN-deficient cells in three independent samples. (B) Normalized intensities expressed as a percentage of the BIP intensity shown in (A) . Actin was used as a loading control. The columns and bar show the mean and standard deviation. Student’s t -test, FXN-138.1 p = 0.008, FXN-138.2 p = 0.023 versus pLKO.1-NT. (C) Quantification of apoptotic cells by flow cytometry. Untreated cells (white bars) and cells treated with thapsigargin (TG, black) were fixed and stained with propidium iodine. The subG1 population was quantified at least in three independent experiments. Bars show mean ± standard deviation. Student’s t -test was applied for statistics. Every cell types were compared between basal condition and with TG: SH-SY5Y p = 0.16, pLKO.1-NT p = 0.042, FXN-138.1 p = 0.032, FXN-138.2 p = 0.001. Comparison after TG treatment between pLKO.1-NT and FXN-138.1 p = 0.050, and FXN-138.2 p = 0.00004. The activation of caspase-3 was tested by western blot (lower panel) using specific antibody in resting conditions (-) and TG-treated cells (+). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
    Figure Legend Snippet: FXN depletion results in increased endoplasmic reticulum (ER) stress. (A) Western blot analysis of ER stress marker BIP in control and FXN-deficient cells in three independent samples. (B) Normalized intensities expressed as a percentage of the BIP intensity shown in (A) . Actin was used as a loading control. The columns and bar show the mean and standard deviation. Student’s t -test, FXN-138.1 p = 0.008, FXN-138.2 p = 0.023 versus pLKO.1-NT. (C) Quantification of apoptotic cells by flow cytometry. Untreated cells (white bars) and cells treated with thapsigargin (TG, black) were fixed and stained with propidium iodine. The subG1 population was quantified at least in three independent experiments. Bars show mean ± standard deviation. Student’s t -test was applied for statistics. Every cell types were compared between basal condition and with TG: SH-SY5Y p = 0.16, pLKO.1-NT p = 0.042, FXN-138.1 p = 0.032, FXN-138.2 p = 0.001. Comparison after TG treatment between pLKO.1-NT and FXN-138.1 p = 0.050, and FXN-138.2 p = 0.00004. The activation of caspase-3 was tested by western blot (lower panel) using specific antibody in resting conditions (-) and TG-treated cells (+). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Techniques Used: Western Blot, Marker, Standard Deviation, Flow Cytometry, Staining, Activation Assay

    20) Product Images from "Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response"

    Article Title: Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.796920

    Constitutional formulae of Tg ( A ), EpoTg ( B ), and thapsigargin analogs in which the butanoyl group of thapsigargin at O-8 is substituted by 12-aminododecanoyl (8ADT; C ) or the 12-amino group of 8ADT is blocked by Boc (Boc-8ADT; D ) or terminates in Leu (Leu-8ADT; E ) or βAsp (βAsp-8ADT; F ).
    Figure Legend Snippet: Constitutional formulae of Tg ( A ), EpoTg ( B ), and thapsigargin analogs in which the butanoyl group of thapsigargin at O-8 is substituted by 12-aminododecanoyl (8ADT; C ) or the 12-amino group of 8ADT is blocked by Boc (Boc-8ADT; D ) or terminates in Leu (Leu-8ADT; E ) or βAsp (βAsp-8ADT; F ).

    Techniques Used:

    21) Product Images from "Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells"

    Article Title: Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers11010083

    Effects of DFMO treatment on store-operated currents (I SOC ) in colon cancer HT29 cells. ( a ) Current–voltage relationships (I–V) of I SOC activated with 1 µM thapsigargin in HT29 colon cancer cells (Red) and NCM460 normal colonic cells (Green) with intracellular medium containing strong Ca 2+ buffer (20 mM EGTA). ( b ) From left to right, I–V relationships for untreated HT29 cells (Red) and HT29 cells exposed to 5 mM DFMO for 6, 24, and 96 h, respectively. Shown in green is I–V relationship of representative current in NCM460 cells. ( c ) Averaged time course of I SOC obtained from HT29 cells, at −80 mV and 80 mV, for control cells (red circles) and cells exposed to 5 mM DFMO for 6, 24, and 96 h, respectively (grey circles, mean ± SEM, n = 7–11). ( d ) Representative I-V relationship and time course of I SOC in HT29 cells treated with 500 µM DFMO for 6 h (mean ± SEM, n = 7). ( e ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1µM thapsigargin for control cells and DFMO-treated cells (mean ± SEM of 7–11 separate experiments, * p
    Figure Legend Snippet: Effects of DFMO treatment on store-operated currents (I SOC ) in colon cancer HT29 cells. ( a ) Current–voltage relationships (I–V) of I SOC activated with 1 µM thapsigargin in HT29 colon cancer cells (Red) and NCM460 normal colonic cells (Green) with intracellular medium containing strong Ca 2+ buffer (20 mM EGTA). ( b ) From left to right, I–V relationships for untreated HT29 cells (Red) and HT29 cells exposed to 5 mM DFMO for 6, 24, and 96 h, respectively. Shown in green is I–V relationship of representative current in NCM460 cells. ( c ) Averaged time course of I SOC obtained from HT29 cells, at −80 mV and 80 mV, for control cells (red circles) and cells exposed to 5 mM DFMO for 6, 24, and 96 h, respectively (grey circles, mean ± SEM, n = 7–11). ( d ) Representative I-V relationship and time course of I SOC in HT29 cells treated with 500 µM DFMO for 6 h (mean ± SEM, n = 7). ( e ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1µM thapsigargin for control cells and DFMO-treated cells (mean ± SEM of 7–11 separate experiments, * p

    Techniques Used:

    Effects of combination DFMO and sulindac on I SOC and SOCE in HT29 cells. ( a ) I–V relationship and ( b ) averaged time course of I SOC HT29 cells exposed to 500 µM DFMO plus 100 µM sulindac for 6 h ( n = 4–12). ( c ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1µM thapsigargin for control cells and cells exposed to DFMO plus sulindac (mean ± SEM of 4–12 separate experiments, * p
    Figure Legend Snippet: Effects of combination DFMO and sulindac on I SOC and SOCE in HT29 cells. ( a ) I–V relationship and ( b ) averaged time course of I SOC HT29 cells exposed to 500 µM DFMO plus 100 µM sulindac for 6 h ( n = 4–12). ( c ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1µM thapsigargin for control cells and cells exposed to DFMO plus sulindac (mean ± SEM of 4–12 separate experiments, * p

    Techniques Used:

    Putrescine reverses the effects of DFMO treatment on I SOC in colon cancer HT29 cells. I SOC were activated with 1 µM thapsigargin in HT29 cells with intracellular medium containing strong Ca 2+ buffer (20 mM EGTA). ( a ) Averaged time course of I SOC obtained from HT29 cells, at −80 mV and 80 mV, for control untreated cells (red circles) and cells exposed for 6 h to 5 mM DFMO plus 200 µM, 500 µM, and 5 mM putrescine, respectively (black circles, mean ± SEM, n = 10–12). ( b ) Representative I–V relationship and time course of I SOC in HT29 cells treated for 6 h with 500 µM DFMO plus 500 µM putrescine (mean ± SEM, n = 10). ( c ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1 µM thapsigargin for control cells and cells exposed to DFMO plus putrescine at different concentrations (mean ± SEM of 10 to 12 separate experiments, * p
    Figure Legend Snippet: Putrescine reverses the effects of DFMO treatment on I SOC in colon cancer HT29 cells. I SOC were activated with 1 µM thapsigargin in HT29 cells with intracellular medium containing strong Ca 2+ buffer (20 mM EGTA). ( a ) Averaged time course of I SOC obtained from HT29 cells, at −80 mV and 80 mV, for control untreated cells (red circles) and cells exposed for 6 h to 5 mM DFMO plus 200 µM, 500 µM, and 5 mM putrescine, respectively (black circles, mean ± SEM, n = 10–12). ( b ) Representative I–V relationship and time course of I SOC in HT29 cells treated for 6 h with 500 µM DFMO plus 500 µM putrescine (mean ± SEM, n = 10). ( c ) Bar graphs are averages of I SOC measured after 5 min of stimulation with 1 µM thapsigargin for control cells and cells exposed to DFMO plus putrescine at different concentrations (mean ± SEM of 10 to 12 separate experiments, * p

    Techniques Used:

    22) Product Images from "Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry"

    Article Title: Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150921

    PCB19 inhibits store-operated cation entry. A , Mn 2+ -induced fura-2 fluorescence quenching was recorded in fura-2/AM-loaded PC12 cells. The fluorescence intensities at 360 nm (F 360 ) was monitored with 1 mM MnCl 2 (arrow), after the preincubation of thapsigargin (TG) with PCB19 or SK F96365 (SK F) in the absence of extracellular free Ca 2+ . B , The changes in time (Δt) during the fluorescence changes (arbitrary units) were quantitatively analyzed with the results in A . C, Ca 2+ store depletion-induced cation influx was measured in PC12 cells with whole-cell patch clamp experiments. Currents were activated following dialysis with 10 mM BAPTA and ramp pulses of membrane potentials from -100 to +100 mV were applied to monitor SOCE current. Typical traces of Ca 2+ store depletion-induced cation influxes with (gray trace) and without (black trace) 50 μM PCB19 are depicted. D, Comparison of average peak store-operated current densities (pA/pF). Number of experiments are depicted in bar graph and each point represents mean ± SEM. ** P
    Figure Legend Snippet: PCB19 inhibits store-operated cation entry. A , Mn 2+ -induced fura-2 fluorescence quenching was recorded in fura-2/AM-loaded PC12 cells. The fluorescence intensities at 360 nm (F 360 ) was monitored with 1 mM MnCl 2 (arrow), after the preincubation of thapsigargin (TG) with PCB19 or SK F96365 (SK F) in the absence of extracellular free Ca 2+ . B , The changes in time (Δt) during the fluorescence changes (arbitrary units) were quantitatively analyzed with the results in A . C, Ca 2+ store depletion-induced cation influx was measured in PC12 cells with whole-cell patch clamp experiments. Currents were activated following dialysis with 10 mM BAPTA and ramp pulses of membrane potentials from -100 to +100 mV were applied to monitor SOCE current. Typical traces of Ca 2+ store depletion-induced cation influxes with (gray trace) and without (black trace) 50 μM PCB19 are depicted. D, Comparison of average peak store-operated current densities (pA/pF). Number of experiments are depicted in bar graph and each point represents mean ± SEM. ** P

    Techniques Used: Fluorescence, Patch Clamp

    PCB19 inhibits thapsigargin-induced SOCE in a manner similar to other SOCE antagonists. A, Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin (TG), then sequentially challenged with 100 μM PCB19 and 30 μM 2-aminoethylphenyl borate (2APB). B, Cells were treated with 1 μM thapsigargin, challenged with 20 μM 2APB, and then treated with 50 μM PCB19. C, Cells were treated with 1 μM thapsigargin, then challenged sequentially with 100 μM PCB19 and 20 μM SK F96365 (SKF). D, Cells were treated with 1 μM thapsigargin, challenged with 20 μM SK F96365, and then treated with 50 μM PCB19. The [Ca 2+ ]i level at point a , b , and c were quantitatively analyzed using calcium traces. Number of experiments are depicted in bar graph and each point represents mean ± SEM.
    Figure Legend Snippet: PCB19 inhibits thapsigargin-induced SOCE in a manner similar to other SOCE antagonists. A, Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin (TG), then sequentially challenged with 100 μM PCB19 and 30 μM 2-aminoethylphenyl borate (2APB). B, Cells were treated with 1 μM thapsigargin, challenged with 20 μM 2APB, and then treated with 50 μM PCB19. C, Cells were treated with 1 μM thapsigargin, then challenged sequentially with 100 μM PCB19 and 20 μM SK F96365 (SKF). D, Cells were treated with 1 μM thapsigargin, challenged with 20 μM SK F96365, and then treated with 50 μM PCB19. The [Ca 2+ ]i level at point a , b , and c were quantitatively analyzed using calcium traces. Number of experiments are depicted in bar graph and each point represents mean ± SEM.

    Techniques Used:

    PCB19 inhibits ionomycin and thapsigargin-induced Ca 2+ influxes. (A, C, E) Fura-2-loaded PC12 cells were incubated in Ca 2+ -free Locke’s solution, challenged with 50 μM PCB19, 1 μM thapsigargin or 300 nM ionomycin, and treated with 2.2 mM CaCl 2 at the indicated time (arrow). (B, D, F) The [Ca 2+ ]i level at point a (Ca 2+ release) and b (Ca 2+ influx) were quantitatively analyzed using calcium traces and expressed as % of controls. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin. ** P
    Figure Legend Snippet: PCB19 inhibits ionomycin and thapsigargin-induced Ca 2+ influxes. (A, C, E) Fura-2-loaded PC12 cells were incubated in Ca 2+ -free Locke’s solution, challenged with 50 μM PCB19, 1 μM thapsigargin or 300 nM ionomycin, and treated with 2.2 mM CaCl 2 at the indicated time (arrow). (B, D, F) The [Ca 2+ ]i level at point a (Ca 2+ release) and b (Ca 2+ influx) were quantitatively analyzed using calcium traces and expressed as % of controls. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin. ** P

    Techniques Used: Incubation

    Ca 2+ influxes stimulated by PCB19 are relatively small compared to those stimulated by intracellular Ca 2+ -mobilizing chemicals. A , Fura-2-loaded PC12 cells were challenged with 50 μM PCB19 in the presence (left) or absence (right) of 2.2 mM extracellular free Ca 2+ . Ca 2+ increases were also monitored upon reintroduction of 2.2 mM CaCl 2 in the condition lacking extracellular Ca 2+ . B and C, Experiments were performed as in ( A ), but with the addition of 1 μM thapsigargin (B) or 300 nM ionomycin (C). D, Peak height of [Ca 2+ ]i increase was monitored and represented as mean ± SEM. E and F, Cells were treated with 300 nM ionomycin in the absence of extracellular free Ca 2+ with (gray trance) or without (black trace) the pretreatment of 50 μM PCB19 for 100 sec. Ca 2+ influx was then measured upon reintroduction of 2.2 mM CaCl 2 (Ca 2+ ) into the extracellular space to monitor the ionomycin-induced Ca 2+ influx. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin.
    Figure Legend Snippet: Ca 2+ influxes stimulated by PCB19 are relatively small compared to those stimulated by intracellular Ca 2+ -mobilizing chemicals. A , Fura-2-loaded PC12 cells were challenged with 50 μM PCB19 in the presence (left) or absence (right) of 2.2 mM extracellular free Ca 2+ . Ca 2+ increases were also monitored upon reintroduction of 2.2 mM CaCl 2 in the condition lacking extracellular Ca 2+ . B and C, Experiments were performed as in ( A ), but with the addition of 1 μM thapsigargin (B) or 300 nM ionomycin (C). D, Peak height of [Ca 2+ ]i increase was monitored and represented as mean ± SEM. E and F, Cells were treated with 300 nM ionomycin in the absence of extracellular free Ca 2+ with (gray trance) or without (black trace) the pretreatment of 50 μM PCB19 for 100 sec. Ca 2+ influx was then measured upon reintroduction of 2.2 mM CaCl 2 (Ca 2+ ) into the extracellular space to monitor the ionomycin-induced Ca 2+ influx. Number of experiments are depicted in bar graph and each point represents mean ± SEM. TG, thapsigargin.

    Techniques Used: Size-exclusion Chromatography

    PCB19 blunts thapsigargin-induced increases in sustained [Ca 2+ ] i levels. A , Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin; 5 minutes later (at the sustained phase), cells were challenged with 50 μM of either PCB4, PCB19, or PCB100. Data presented include typical Ca 2+ traces from more than five independent experiments. B , Concentration-dependent effects of PCBs on thapsigargin-induced SOCE. Decreases in Ca 2+ levels were monitored upon stimulation with various concentrations of PCB4 (filled triangles), PCB19 (filled squares), PCB50 (blank circles), and PCB100 (blank triangles). Net decreases in [Ca 2+ ] i are expressed as % of controls (thapsigargin-induced Ca 2+ levels without PCB19 treatment). Each point shown was obtained from triplicate experiments and represents the mean ± SEM. TG, thapsigargin.
    Figure Legend Snippet: PCB19 blunts thapsigargin-induced increases in sustained [Ca 2+ ] i levels. A , Fura-2-loaded PC12 cells were treated with 1 μM thapsigargin; 5 minutes later (at the sustained phase), cells were challenged with 50 μM of either PCB4, PCB19, or PCB100. Data presented include typical Ca 2+ traces from more than five independent experiments. B , Concentration-dependent effects of PCBs on thapsigargin-induced SOCE. Decreases in Ca 2+ levels were monitored upon stimulation with various concentrations of PCB4 (filled triangles), PCB19 (filled squares), PCB50 (blank circles), and PCB100 (blank triangles). Net decreases in [Ca 2+ ] i are expressed as % of controls (thapsigargin-induced Ca 2+ levels without PCB19 treatment). Each point shown was obtained from triplicate experiments and represents the mean ± SEM. TG, thapsigargin.

    Techniques Used: Concentration Assay

    23) Product Images from "Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum"

    Article Title: Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085351

    The slow Ca 2+ oscillations in both putative-neurons and astrocytes were mainly due to Ca 2+ release from the intracellular Ca 2+ store via the IP 3 receptor. A–D, Typical time courses of the slow Ca 2+ oscillations during the administration of 10 µM CNQX and 50 µM AP5 (CNQX + AP5), 1 µM TTX, 2 µM thapsigargin (Thapsi), and 100 µM 2-APB in putative-neurons and astrocytes. Horizontal bars under the time courses indicate the application period of the agents. Scale bar, 200 s, µR = 0.02. E, F, Transient rates of the slow Ca 2+ oscillations during the administration of various pharmacological agents in putative-neurons (E) and astrocytes (F). The transient rates of the slow Ca 2+ oscillations are normalized by the transient rates under control conditions. The number of cells recorded is shown above each bar graph. ****p
    Figure Legend Snippet: The slow Ca 2+ oscillations in both putative-neurons and astrocytes were mainly due to Ca 2+ release from the intracellular Ca 2+ store via the IP 3 receptor. A–D, Typical time courses of the slow Ca 2+ oscillations during the administration of 10 µM CNQX and 50 µM AP5 (CNQX + AP5), 1 µM TTX, 2 µM thapsigargin (Thapsi), and 100 µM 2-APB in putative-neurons and astrocytes. Horizontal bars under the time courses indicate the application period of the agents. Scale bar, 200 s, µR = 0.02. E, F, Transient rates of the slow Ca 2+ oscillations during the administration of various pharmacological agents in putative-neurons (E) and astrocytes (F). The transient rates of the slow Ca 2+ oscillations are normalized by the transient rates under control conditions. The number of cells recorded is shown above each bar graph. ****p

    Techniques Used:

    24) Product Images from "Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca2+ current ICRAC"

    Article Title: Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca2+ current ICRAC

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.23.6401

    Fig. 2. Respiring mitochondria increase the size of I CRAC in weak but not strong buffer. ( A ) Time-course of I CRAC in weak buffer in the absence (control) and presence of the mitochondrial cocktail. ( B ) I CRAC is significantly bigger in the presence of cocktail compared with control. ( C ) Pre-treatment with antimycin A and oligomycin prevents the enhancing effect of cocktail. The experiments were carried out on cells from the same preparations using the same solutions. Cells were incubated for at least 20 min in 5 µg/ml antimycin A and 0.5 µg/ml oligomycin. ( D ) I–V relationship for a cell dialysed with cocktail when I CRAC had peaked. ( E ) A plot of current amplitude against % of responding cells in weak and strong buffer in the absence and presence of cocktail. ( F ) Extent of rapid inactivation in the absence (control) and presence of cocktail. For all panels, control refers to InsP 3 + thapsigargin + 0.1 mM EGTA + ATP.
    Figure Legend Snippet: Fig. 2. Respiring mitochondria increase the size of I CRAC in weak but not strong buffer. ( A ) Time-course of I CRAC in weak buffer in the absence (control) and presence of the mitochondrial cocktail. ( B ) I CRAC is significantly bigger in the presence of cocktail compared with control. ( C ) Pre-treatment with antimycin A and oligomycin prevents the enhancing effect of cocktail. The experiments were carried out on cells from the same preparations using the same solutions. Cells were incubated for at least 20 min in 5 µg/ml antimycin A and 0.5 µg/ml oligomycin. ( D ) I–V relationship for a cell dialysed with cocktail when I CRAC had peaked. ( E ) A plot of current amplitude against % of responding cells in weak and strong buffer in the absence and presence of cocktail. ( F ) Extent of rapid inactivation in the absence (control) and presence of cocktail. For all panels, control refers to InsP 3 + thapsigargin + 0.1 mM EGTA + ATP.

    Techniques Used: Incubation

    Fig. 4. I CRAC activates to InsP 3 alone in weak buffer provided mitochondria are active. ( A ) Time-course of I CRAC for a cell dialysed with InsP 3 + 0.1 mM EGTA + ATP (control) and then for one supplemented with cocktail. No thapsigargin was present. ( B ) I–V relationship from voltage ramps is shown (taken when after 60 s). ( C ) Bar chart depicting the enhancing effect of cocktail, and that the mitochondrial inhibitors antimycin A (+ oligomycin) and ruthenium red prevent the effects of cocktail. Control + cAMP or GTP did not mimic the effects of cocktail. Again, thapsigargin was not present. ( D ) Plot of amplitude of I CRAC versus % responding cells for control and cocktail-treated cells (no thapsigargin present). Only the amplitudes of responding cells were analysed. ( E ) τ-activation versus amplitude of I CRAC is plotted for cocktail-treated cells in weak buffer (no thapsigargin) and strong buffer (InsP 3 + thapsigargin + 10 mM EGTA + ATP). Note that in weak buffer without thapsigargin, I CRAC was still sub-maximal in spite of cocktail. This reflects some SERCA-dependent refilling, because the current was larger in weak buffer when thapsigargin was included.
    Figure Legend Snippet: Fig. 4. I CRAC activates to InsP 3 alone in weak buffer provided mitochondria are active. ( A ) Time-course of I CRAC for a cell dialysed with InsP 3 + 0.1 mM EGTA + ATP (control) and then for one supplemented with cocktail. No thapsigargin was present. ( B ) I–V relationship from voltage ramps is shown (taken when after 60 s). ( C ) Bar chart depicting the enhancing effect of cocktail, and that the mitochondrial inhibitors antimycin A (+ oligomycin) and ruthenium red prevent the effects of cocktail. Control + cAMP or GTP did not mimic the effects of cocktail. Again, thapsigargin was not present. ( D ) Plot of amplitude of I CRAC versus % responding cells for control and cocktail-treated cells (no thapsigargin present). Only the amplitudes of responding cells were analysed. ( E ) τ-activation versus amplitude of I CRAC is plotted for cocktail-treated cells in weak buffer (no thapsigargin) and strong buffer (InsP 3 + thapsigargin + 10 mM EGTA + ATP). Note that in weak buffer without thapsigargin, I CRAC was still sub-maximal in spite of cocktail. This reflects some SERCA-dependent refilling, because the current was larger in weak buffer when thapsigargin was included.

    Techniques Used: Activation Assay

    Fig. 5. Cartoon summary of mitochondrial role in I CRAC in weak (physiological) intracellular Ca 2+ buffer. ( A ) Shows the resting state, where I CRAC is not functioning. Stores are full and any Ca 2+ that leaks from the stores is re-sequestrated by the SERCA pumps. ( B ) Following an increase in the levels of the second messenger InsP 3 in the absence of active mitochondrial Ca 2+ uptake, Ca 2+ is released from the stores. However, the SERCA pumps are able to re-sequestrate sufficient Ca 2+ to prevent the threshold for macroscopic activation of I CRAC from being reached. Only a very small fraction of CRAC channels are activated (undetectable in whole-cell mode). Furthermore, the rise in cytosolic Ca 2+ results in strong inactivation of I CRAC through Ca 2+ -dependent slow inactivation. ( C ) In the presence of respiring mitochondria, InsP 3 activates macroscopic I CRAC . Ca 2+ released from the stores by InsP 3 is taken up by mitochondria through a ruthenium red-sensitive uniporter. This reduces the amount of Ca 2+ available to the SERCA pumps and in the vicinity of open InsP 3 receptors, such that the stores are depleted sufficiently for macroscopic I CRAC to activate (less refilling by SERCA pumps and less inactivation of InsP 3 receptors). I CRAC now activates. Some refilling does occur because inclusion of thapsigargin enhances the size of the current. Furthermore, mitochondrial Ca 2+ buffering reduces the rate and extent of Ca 2+ -dependent slow inactivation, thereby increasing the size and duration of the current. ( D ) A simplified gating scheme for CRAC channels summarizing the role of mitochondrial Ca 2+ buffering. Mitochondria facilitate opening (Closed to Open transition) whilst simultaneously reducing inactivation (Open to Inactivated transition). In this way, mitochondria have a much larger impact on I CRAC than through either transition alone.
    Figure Legend Snippet: Fig. 5. Cartoon summary of mitochondrial role in I CRAC in weak (physiological) intracellular Ca 2+ buffer. ( A ) Shows the resting state, where I CRAC is not functioning. Stores are full and any Ca 2+ that leaks from the stores is re-sequestrated by the SERCA pumps. ( B ) Following an increase in the levels of the second messenger InsP 3 in the absence of active mitochondrial Ca 2+ uptake, Ca 2+ is released from the stores. However, the SERCA pumps are able to re-sequestrate sufficient Ca 2+ to prevent the threshold for macroscopic activation of I CRAC from being reached. Only a very small fraction of CRAC channels are activated (undetectable in whole-cell mode). Furthermore, the rise in cytosolic Ca 2+ results in strong inactivation of I CRAC through Ca 2+ -dependent slow inactivation. ( C ) In the presence of respiring mitochondria, InsP 3 activates macroscopic I CRAC . Ca 2+ released from the stores by InsP 3 is taken up by mitochondria through a ruthenium red-sensitive uniporter. This reduces the amount of Ca 2+ available to the SERCA pumps and in the vicinity of open InsP 3 receptors, such that the stores are depleted sufficiently for macroscopic I CRAC to activate (less refilling by SERCA pumps and less inactivation of InsP 3 receptors). I CRAC now activates. Some refilling does occur because inclusion of thapsigargin enhances the size of the current. Furthermore, mitochondrial Ca 2+ buffering reduces the rate and extent of Ca 2+ -dependent slow inactivation, thereby increasing the size and duration of the current. ( D ) A simplified gating scheme for CRAC channels summarizing the role of mitochondrial Ca 2+ buffering. Mitochondria facilitate opening (Closed to Open transition) whilst simultaneously reducing inactivation (Open to Inactivated transition). In this way, mitochondria have a much larger impact on I CRAC than through either transition alone.

    Techniques Used: Activation Assay

    25) Product Images from "Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1"

    Article Title: Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201506022

    Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM thapsigargin in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P
    Figure Legend Snippet: Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM thapsigargin in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P

    Techniques Used: Expressing, Fluorescence, Translocation Assay

    σ1R accompanies STIM1 to ER–PM junctions after store depletion. (A) Immunoblot of lysates from wild-type (WT) HEK, HEK-σ1R, or HeLa cells. The same amount of protein was loaded in each lane. (B) Confocal images of unstimulated HeLa cells transiently transfected with σ1R-EGFP and mCh-STIM1. Bar, 10 µm. (Right) Enlargement of the boxed area. Bar, 2.5 µm. (C and D) TIRF images of HeLa cells expressing mCh-STIM1 (C, top), σ1R-EGFP (C, bottom), or both (D) before and 10 min after addition of 5 µM thapsigargin in Ca 2+ -free HBS. (E, top) Traces show time courses of the fluorescence changes (F/F 0 ) within the TIRF field after addition of thapsigargin (mean values for 30 puncta for each or size-matched regions of interest for σ1R alone). (E, bottom) Summary results show changes in mCh fluorescence (normalized to maximal intensity) after store depletion in cells with and without σ1R ( n = 87). (F and G) TIRF images of HeLa cells expressing Orai1-EGFP and σ1R-mKate either with (F) or without HA-STIM1 (G). Bars (C, D, F, and G), 10 µm. (H) Confocal images of HeLa cells expressing σ1R-EGFP, HA-STIM1, and Orai1-Myc, immunostained after treatment with 5 µM thapsigargin. Boxed areas in the left panels (bar, 5 µm) are enlarged on the right (bar, 2 µm). Arrowheads show colocalization of all three proteins as white puncta at the PM. (I) Summary results ( n = 8) show Mander’s overlap coefficient for colocalization of the indicated pairs of proteins in cells expressing only those tagged proteins or with σ1R-EGFP or HA-STIM1, as indicated, with and without thapsigargin treatment. **, P
    Figure Legend Snippet: σ1R accompanies STIM1 to ER–PM junctions after store depletion. (A) Immunoblot of lysates from wild-type (WT) HEK, HEK-σ1R, or HeLa cells. The same amount of protein was loaded in each lane. (B) Confocal images of unstimulated HeLa cells transiently transfected with σ1R-EGFP and mCh-STIM1. Bar, 10 µm. (Right) Enlargement of the boxed area. Bar, 2.5 µm. (C and D) TIRF images of HeLa cells expressing mCh-STIM1 (C, top), σ1R-EGFP (C, bottom), or both (D) before and 10 min after addition of 5 µM thapsigargin in Ca 2+ -free HBS. (E, top) Traces show time courses of the fluorescence changes (F/F 0 ) within the TIRF field after addition of thapsigargin (mean values for 30 puncta for each or size-matched regions of interest for σ1R alone). (E, bottom) Summary results show changes in mCh fluorescence (normalized to maximal intensity) after store depletion in cells with and without σ1R ( n = 87). (F and G) TIRF images of HeLa cells expressing Orai1-EGFP and σ1R-mKate either with (F) or without HA-STIM1 (G). Bars (C, D, F, and G), 10 µm. (H) Confocal images of HeLa cells expressing σ1R-EGFP, HA-STIM1, and Orai1-Myc, immunostained after treatment with 5 µM thapsigargin. Boxed areas in the left panels (bar, 5 µm) are enlarged on the right (bar, 2 µm). Arrowheads show colocalization of all three proteins as white puncta at the PM. (I) Summary results ( n = 8) show Mander’s overlap coefficient for colocalization of the indicated pairs of proteins in cells expressing only those tagged proteins or with σ1R-EGFP or HA-STIM1, as indicated, with and without thapsigargin treatment. **, P

    Techniques Used: Transfection, Expressing, Fluorescence

    Ligands of σ1R modulate SOCE. (A–F) Populations of cells were treated with 25 µM (+)SKF10047 or 10 µM BD1047 before removal of extracellular Ca 2+ , addition of 5 µM thapsigargin, and then restoration of extracellular 4 mM Ca 2+ to CHO (A and B), HEK-σ1R (C and D), or wild-type HEK cells (E and F). Summary results (B, D, and F) show peak increases in [Ca 2+ ] c after restoration of extracellular Ca 2+ . The color codes in A apply to all panels (A–F). (G) Representative immunoblot from CHO cells transfected with control plasmid or plasmid encoding siRNA for σ1R (siσ1R). (H) Summary results show band intensities for the indicated proteins normalized to those from cells treated with control plasmid. (I) Ca 2+ signals evoked by addition of thapsigargin in Ca 2+ -free HBS and then restoration of extracellular Ca 2+ in CHO cells treated with siσ1R or control plasmid. (J) Summary shows peak [Ca 2+ ] c after restoration of extracellular Ca 2+ to thapsigargin-treated CHO cells treated with siσ1R or control plasmid. Cells were pretreated with 25 µM (+)SKF10047 or 10 µM BD1047, as indicated. (K and L) Effects of siσ1R or control plasmid and pretreatment with σ1R ligands on the Ca 2+ signals evoked by 5 µM ionomycin in Ca 2+ -free HBS. Typical traces (K) and summary results (L) are shown. Legends for L are the same as J. All summary results show mean ± SEM. n = 3. *, P
    Figure Legend Snippet: Ligands of σ1R modulate SOCE. (A–F) Populations of cells were treated with 25 µM (+)SKF10047 or 10 µM BD1047 before removal of extracellular Ca 2+ , addition of 5 µM thapsigargin, and then restoration of extracellular 4 mM Ca 2+ to CHO (A and B), HEK-σ1R (C and D), or wild-type HEK cells (E and F). Summary results (B, D, and F) show peak increases in [Ca 2+ ] c after restoration of extracellular Ca 2+ . The color codes in A apply to all panels (A–F). (G) Representative immunoblot from CHO cells transfected with control plasmid or plasmid encoding siRNA for σ1R (siσ1R). (H) Summary results show band intensities for the indicated proteins normalized to those from cells treated with control plasmid. (I) Ca 2+ signals evoked by addition of thapsigargin in Ca 2+ -free HBS and then restoration of extracellular Ca 2+ in CHO cells treated with siσ1R or control plasmid. (J) Summary shows peak [Ca 2+ ] c after restoration of extracellular Ca 2+ to thapsigargin-treated CHO cells treated with siσ1R or control plasmid. Cells were pretreated with 25 µM (+)SKF10047 or 10 µM BD1047, as indicated. (K and L) Effects of siσ1R or control plasmid and pretreatment with σ1R ligands on the Ca 2+ signals evoked by 5 µM ionomycin in Ca 2+ -free HBS. Typical traces (K) and summary results (L) are shown. Legends for L are the same as J. All summary results show mean ± SEM. n = 3. *, P

    Techniques Used: Transfection, Plasmid Preparation

    Inhibition of SOCE by σ1R. (A) Ca 2+ signals recorded from populations of fluo 4–loaded HEK cells transiently transfected with Orai1 E106Q , STIM1 and Orai1, or mock transfected (control). Cells were stimulated with 5 µM thapsigargin in Ca 2+ -free HBS before restoration of extracellular Ca 2+ (final free [Ca 2+ ], 4 mM). Results show mean responses from six replicates. (B) Summary results ( n = 3) show peak increases in [Ca 2+ ] c evoked by thapsigargin (Ca 2+ release) and Ca 2+ restoration (SOCE). (C) Typical immunoblot of σ1R, STIM1, Orai1, and β-actin from 20 µg of solubilized protein from wild-type (WT) HEK and HEK-σ1R cells. (D) Ca 2+ signals evoked by thapsigargin in Ca 2+ -free HBS and after restoration of extracellular Ca 2+ to wild-type and HEK-σ1R cells. (E) Summary shows responses to thapsigargin (Ca 2+ release) and SOCE detected after restoring Ca 2+ 10 or 20 min after thapsigargin ( n = 6). (F) Responses from single fura 2–loaded HEK cells show fluorescence ratios (F 340 /F 380 ) after stimulation with 5 µM thapsigargin and restoration of 4 mM extracellular Ca 2+ . n = 3, each with ∼45 cells. (G) Ca 2+ contents of the intracellular stores determined by measuring [Ca 2+ ] c after addition of 5 µM ionomycin in Ca 2+ -free HBS before or 10 min after treatment with thapsigargin. (H) Summary results ( n = 6). (I) Ca 2+ release and SOCE evoked by 100 µM carbachol and 100 µM ATP. (J) Summary results ( n = 6). *, P
    Figure Legend Snippet: Inhibition of SOCE by σ1R. (A) Ca 2+ signals recorded from populations of fluo 4–loaded HEK cells transiently transfected with Orai1 E106Q , STIM1 and Orai1, or mock transfected (control). Cells were stimulated with 5 µM thapsigargin in Ca 2+ -free HBS before restoration of extracellular Ca 2+ (final free [Ca 2+ ], 4 mM). Results show mean responses from six replicates. (B) Summary results ( n = 3) show peak increases in [Ca 2+ ] c evoked by thapsigargin (Ca 2+ release) and Ca 2+ restoration (SOCE). (C) Typical immunoblot of σ1R, STIM1, Orai1, and β-actin from 20 µg of solubilized protein from wild-type (WT) HEK and HEK-σ1R cells. (D) Ca 2+ signals evoked by thapsigargin in Ca 2+ -free HBS and after restoration of extracellular Ca 2+ to wild-type and HEK-σ1R cells. (E) Summary shows responses to thapsigargin (Ca 2+ release) and SOCE detected after restoring Ca 2+ 10 or 20 min after thapsigargin ( n = 6). (F) Responses from single fura 2–loaded HEK cells show fluorescence ratios (F 340 /F 380 ) after stimulation with 5 µM thapsigargin and restoration of 4 mM extracellular Ca 2+ . n = 3, each with ∼45 cells. (G) Ca 2+ contents of the intracellular stores determined by measuring [Ca 2+ ] c after addition of 5 µM ionomycin in Ca 2+ -free HBS before or 10 min after treatment with thapsigargin. (H) Summary results ( n = 6). (I) Ca 2+ release and SOCE evoked by 100 µM carbachol and 100 µM ATP. (J) Summary results ( n = 6). *, P

    Techniques Used: Inhibition, Transfection, Fluorescence

    STIM1, Orai1, and σ1R interact within a macromolecular complex at the PM. (A) HEK cells expressing σ1R-FLAG alone or with Orai1-Myc or Orai1-Myc and HA-STIM1 were treated with thapsigargin (5 µM for 30 min in Ca 2+ -free HBS), and then the cell surface was biotinylated. The representative immunoblot shows the inputs and the proteins detected after purification with avidin beads. Input lanes were loaded with 10 µl of the 500-µl sample, and surface biotinylation lanes were loaded with 10 µl of the 50-µl eluate. (B) Summary shows the amounts of σ1R-FLAG detected in the avidin pull-downs (normalized to cells expressing only σ1R-FLAG). (C) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were cell surface biotinylated before sequential purification by elution from avidin-agarose with biotin and then from anti-Myc­–agarose with Myc peptide. The immunoblot (anti-HA, anti-FLAG, anti-Myc, and anti–β-actin) shows the input and the two eluates. Input lanes were loaded with 10 µl of the 500-µl sample and elution lanes with 10 µl of the 50-µl eluate. (D) Summary shows the amounts of HA-STIM1 detected in the avidin (biotin elution) and anti-Myc pull-downs (normalized to Orai1-Myc pull-down in each condition). (E) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were immunoprecipitated (IP) with anti-HA antibody. (F) Peak [Ca 2+ ] c signals evoked by SOCE were recorded from HEK or HEK-σ1R cells after treatment with thapsigargin (5 µM in Ca 2+ -free HBS for 10 min) and then restoration of 4 mM extracellular Ca 2+ . The effects of transiently overexpressing STIM1 or Orai1 are shown. WT, wild type. (G) The Ca 2+ contents of the intracellular stores of the same cells were measured by recording peak increases in [Ca 2+ ] c from cells exposed to ionomycin (5 µM in Ca 2+ -free HBS). Results (B, D, F, and G) are mean ± SEM. n = 3. *, P
    Figure Legend Snippet: STIM1, Orai1, and σ1R interact within a macromolecular complex at the PM. (A) HEK cells expressing σ1R-FLAG alone or with Orai1-Myc or Orai1-Myc and HA-STIM1 were treated with thapsigargin (5 µM for 30 min in Ca 2+ -free HBS), and then the cell surface was biotinylated. The representative immunoblot shows the inputs and the proteins detected after purification with avidin beads. Input lanes were loaded with 10 µl of the 500-µl sample, and surface biotinylation lanes were loaded with 10 µl of the 50-µl eluate. (B) Summary shows the amounts of σ1R-FLAG detected in the avidin pull-downs (normalized to cells expressing only σ1R-FLAG). (C) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were cell surface biotinylated before sequential purification by elution from avidin-agarose with biotin and then from anti-Myc­–agarose with Myc peptide. The immunoblot (anti-HA, anti-FLAG, anti-Myc, and anti–β-actin) shows the input and the two eluates. Input lanes were loaded with 10 µl of the 500-µl sample and elution lanes with 10 µl of the 50-µl eluate. (D) Summary shows the amounts of HA-STIM1 detected in the avidin (biotin elution) and anti-Myc pull-downs (normalized to Orai1-Myc pull-down in each condition). (E) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were immunoprecipitated (IP) with anti-HA antibody. (F) Peak [Ca 2+ ] c signals evoked by SOCE were recorded from HEK or HEK-σ1R cells after treatment with thapsigargin (5 µM in Ca 2+ -free HBS for 10 min) and then restoration of 4 mM extracellular Ca 2+ . The effects of transiently overexpressing STIM1 or Orai1 are shown. WT, wild type. (G) The Ca 2+ contents of the intracellular stores of the same cells were measured by recording peak increases in [Ca 2+ ] c from cells exposed to ionomycin (5 µM in Ca 2+ -free HBS). Results (B, D, F, and G) are mean ± SEM. n = 3. *, P

    Techniques Used: Expressing, Purification, Avidin-Biotin Assay, Immunoprecipitation

    26) Product Images from "Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells"

    Article Title: Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12239

    PGE 2 inhibits histamine-evoked Ca 2+ release. (A) Ca 2+ signals evoked by histamine (100 μM, bar) alone or with PGE 2 (10 μM, added 5 min before and then with histamine). Results, means ± SEM from three wells on a single plate, are typical of results from four independent plates. (B) Effect of PGE 2 (10 μM) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± SEM from seven independent plates, each with one to three wells. (C) Effect of PGE 2 on the sustained Ca 2+ signals evoked by histamine. Results are means ± SEM from 11 independent plates, each with one to three wells. (D) Effect of the indicated concentrations of PGE 2 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (3 μM). Results are means ± SEM from 15 independent plates, with one to three wells analysed from each. (B–D) Ct denotes control. Similar results from ASMC isolated from different patients are shown in Supporting Information Figure S1 . (E) Effects of pretreatment with PGE 2 (10 μM, 5 min) on the peak Ca 2+ signals evoked by subsequent addition of thapsigargin (1 μM), cyclopiazonic acid (10 μM) or ionomycin (1 μM) to ASMC in Ca 2+ -free HBS. Results (as percentages of the responses obtained without PGE 2 ) are means ± SEM from three independent plates, with seven wells analysed on each.
    Figure Legend Snippet: PGE 2 inhibits histamine-evoked Ca 2+ release. (A) Ca 2+ signals evoked by histamine (100 μM, bar) alone or with PGE 2 (10 μM, added 5 min before and then with histamine). Results, means ± SEM from three wells on a single plate, are typical of results from four independent plates. (B) Effect of PGE 2 (10 μM) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± SEM from seven independent plates, each with one to three wells. (C) Effect of PGE 2 on the sustained Ca 2+ signals evoked by histamine. Results are means ± SEM from 11 independent plates, each with one to three wells. (D) Effect of the indicated concentrations of PGE 2 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (3 μM). Results are means ± SEM from 15 independent plates, with one to three wells analysed from each. (B–D) Ct denotes control. Similar results from ASMC isolated from different patients are shown in Supporting Information Figure S1 . (E) Effects of pretreatment with PGE 2 (10 μM, 5 min) on the peak Ca 2+ signals evoked by subsequent addition of thapsigargin (1 μM), cyclopiazonic acid (10 μM) or ionomycin (1 μM) to ASMC in Ca 2+ -free HBS. Results (as percentages of the responses obtained without PGE 2 ) are means ± SEM from three independent plates, with seven wells analysed on each.

    Techniques Used: Isolation

    27) Product Images from "TALK-1 channels control β cell endoplasmic reticulum Ca2+ homeostasis"

    Article Title: TALK-1 channels control β cell endoplasmic reticulum Ca2+ homeostasis

    Journal: Science signaling

    doi: 10.1126/scisignal.aan2883

    TALK-1 regulates Ca 2+ ER handling during plasma membrane Ca 2+ influx in β-cells ( A ) Intracellular Ca 2+ oscillations in response to pulses of 45 mM K + (K45) for 40 seconds in the presence or absence of thapsigargin (1.25 µM). Recordings were performed in the presence of 11 mM glucose (G), 2.5 mM Ca 2+ , and 125 µM diazoxide (Dz). ( B ) Subtraction of the thapsigargin-treated trace from the control trace in A reveals the kinetics of Ca 2+ ER uptake and release. ( C ) Quantification of average Ca 2+ ER uptake and release in WT and TALK-1 KO β-cells ( N = 3 mice per genotype). ( D ) Effect of CPA on glucose-stimulated Ca 2+ influx in WT and KO islets. ( E ) Area under the curve (AUC) analysis of glucose-stimulated Ca 2+ influx for periods corresponding to low glucose (2G), high glucose (11G), and CPA (11G + CPA) ( N = 49 WT and 53 TALK-1 KO islets). Statistical significance was determined by Student’s t -test; * P
    Figure Legend Snippet: TALK-1 regulates Ca 2+ ER handling during plasma membrane Ca 2+ influx in β-cells ( A ) Intracellular Ca 2+ oscillations in response to pulses of 45 mM K + (K45) for 40 seconds in the presence or absence of thapsigargin (1.25 µM). Recordings were performed in the presence of 11 mM glucose (G), 2.5 mM Ca 2+ , and 125 µM diazoxide (Dz). ( B ) Subtraction of the thapsigargin-treated trace from the control trace in A reveals the kinetics of Ca 2+ ER uptake and release. ( C ) Quantification of average Ca 2+ ER uptake and release in WT and TALK-1 KO β-cells ( N = 3 mice per genotype). ( D ) Effect of CPA on glucose-stimulated Ca 2+ influx in WT and KO islets. ( E ) Area under the curve (AUC) analysis of glucose-stimulated Ca 2+ influx for periods corresponding to low glucose (2G), high glucose (11G), and CPA (11G + CPA) ( N = 49 WT and 53 TALK-1 KO islets). Statistical significance was determined by Student’s t -test; * P

    Techniques Used: Mouse Assay

    28) Product Images from "Contractile effect of tachykinins on rabbit small intestine"

    Article Title: Contractile effect of tachykinins on rabbit small intestine

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2010.227

    Effect of Ca 2+ -free solutions containing 0.5 mmol/L EGTA (0Ca), verapamil (V, 100 nmol/L), thapsigargin (T, 100 nmol/L), ryanodine (R, 100 nmol/L), staurosporine (St, 100 nmol/L), and U 73122 (U, 100 nmol/L) on contractions caused by SP (100 nmol/L) in longitudinal (A) and circular (B) smooth muscle of rabbit duodenum, jejunum, and ileum. Columns indicate the mean values of integrated mechanical activity (% of SP), and vertical bars indicate SEM. b P
    Figure Legend Snippet: Effect of Ca 2+ -free solutions containing 0.5 mmol/L EGTA (0Ca), verapamil (V, 100 nmol/L), thapsigargin (T, 100 nmol/L), ryanodine (R, 100 nmol/L), staurosporine (St, 100 nmol/L), and U 73122 (U, 100 nmol/L) on contractions caused by SP (100 nmol/L) in longitudinal (A) and circular (B) smooth muscle of rabbit duodenum, jejunum, and ileum. Columns indicate the mean values of integrated mechanical activity (% of SP), and vertical bars indicate SEM. b P

    Techniques Used: Activity Assay

    29) Product Images from "Nifedipine facilitates neurotransmitter release independently of calcium channels"

    Article Title: Nifedipine facilitates neurotransmitter release independently of calcium channels

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

    doi: 10.1073/pnas.0936131100

    Nifedipine action is independent of calcium. ( A ) Sample current traces of three different neurons, treated with Cd 2+ , thapsigargin, or BAPTA-AM ( Upper ) and the effect of nifedipine on these treated cells ( Lower ). ( B ) Sample evoked EPSCs of three different neurons, showing the effect of Cd 2+ , thapsigargin, or BAPTA-AM. ( C ) Summary of the effect of Cd 2+ , thapsigargin, or BAPTA-AM on nifedipine-induced mEPSC frequency. In these cells, nifedipine was applied first. ( D ) Summary of the effect of Cd 2+ , thapsigargin, or BAPTA-AM on evoked EPSC amplitude. *, P
    Figure Legend Snippet: Nifedipine action is independent of calcium. ( A ) Sample current traces of three different neurons, treated with Cd 2+ , thapsigargin, or BAPTA-AM ( Upper ) and the effect of nifedipine on these treated cells ( Lower ). ( B ) Sample evoked EPSCs of three different neurons, showing the effect of Cd 2+ , thapsigargin, or BAPTA-AM. ( C ) Summary of the effect of Cd 2+ , thapsigargin, or BAPTA-AM on nifedipine-induced mEPSC frequency. In these cells, nifedipine was applied first. ( D ) Summary of the effect of Cd 2+ , thapsigargin, or BAPTA-AM on evoked EPSC amplitude. *, P

    Techniques Used:

    30) Product Images from "Biallelic loss-of-function OBSCN variants predispose individuals to severe, recurrent rhabdomyolysis"

    Article Title: Biallelic loss-of-function OBSCN variants predispose individuals to severe, recurrent rhabdomyolysis

    Journal: bioRxiv

    doi: 10.1101/2021.06.04.447044

    Studies from patient (UK1) myoblasts show aberrant Ca 2+ flux and increased cell death. (A) SR morphology is not altered in patient myoblasts when compared to control myoblasts as shown by immunostaining with anti-calnexin antibody and confocal analysis. ( B ) Total SR content and the morphological parameters measured (length, sphericity) are similar in healthy control (CTRL) and patient myoblasts. (C) Representative SR Ca 2+ content measurements in myoblasts from a healthy control (CTRL, left panel) and patient (right panel) in control (GM, black traces) and EBSS medium for 2 hours (red traces). ( D ) SR Ca 2+ content was assessed from the area under the curves (AUC) after thapsigargin addition. Horizontal bar, 0 seconds; vertical bars F/Fmax 0.1 (arbitrary units). Histograms summarising area under the curve (AUC) in control (GM) and EBSS media. Data from 12 and six individual wells for CTRL and patient respectively obtained from two independent experiments corresponding to a decrease of 33±2 % (CTRL) and 69±6 % (Patient) of SR Ca 2+ contents in EBSS medium. ( E ) Apoptosis was assessed from purple events representing caspase 3/7 positive cells normalised to cell number. Patient myoblasts show higher levels of apoptosis (2.3-fold) as detected by caspase 3/7 expression when compare to CRTL myoblasts. ( F ) Quantification of caspase 3/7 expression in control and patient myoblasts. Results of one representative experiment out of two independent experiments.
    Figure Legend Snippet: Studies from patient (UK1) myoblasts show aberrant Ca 2+ flux and increased cell death. (A) SR morphology is not altered in patient myoblasts when compared to control myoblasts as shown by immunostaining with anti-calnexin antibody and confocal analysis. ( B ) Total SR content and the morphological parameters measured (length, sphericity) are similar in healthy control (CTRL) and patient myoblasts. (C) Representative SR Ca 2+ content measurements in myoblasts from a healthy control (CTRL, left panel) and patient (right panel) in control (GM, black traces) and EBSS medium for 2 hours (red traces). ( D ) SR Ca 2+ content was assessed from the area under the curves (AUC) after thapsigargin addition. Horizontal bar, 0 seconds; vertical bars F/Fmax 0.1 (arbitrary units). Histograms summarising area under the curve (AUC) in control (GM) and EBSS media. Data from 12 and six individual wells for CTRL and patient respectively obtained from two independent experiments corresponding to a decrease of 33±2 % (CTRL) and 69±6 % (Patient) of SR Ca 2+ contents in EBSS medium. ( E ) Apoptosis was assessed from purple events representing caspase 3/7 positive cells normalised to cell number. Patient myoblasts show higher levels of apoptosis (2.3-fold) as detected by caspase 3/7 expression when compare to CRTL myoblasts. ( F ) Quantification of caspase 3/7 expression in control and patient myoblasts. Results of one representative experiment out of two independent experiments.

    Techniques Used: Immunostaining, Expressing

    31) Product Images from "Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ influx and subsequent initiation of sperm motility in the common carp"

    Article Title: Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ influx and subsequent initiation of sperm motility in the common carp

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

    doi:

    Effect of the Ca 2+ mobilizer thapsigargin on the intracellular Ca 2+ concentration of the quiescent cells. Sperm were loaded with calcium green1-AM as described in Materials and Methods and suspended in NoCaFPS (closed circle) or FPS containing 100 nM Ca 2+ (open diamond) or 1,000 nM Ca 2+ (open circle), and 50 μM thapsigargin and fluorescence from the cells was monitored for 10 min. Control cells were suspended in FPS containing 100 nM Ca 2+ (closed circle), and at t = 10 min, 5 μM A 23187 calcium ionophore was added to the suspension and the fluorescence from the sperm cells was measured. When 5 μM A 23187 was added to the sperm cells containing different calcium concentrations and thapsigargin (arrow), the intracellular calcium concentration reached that of the extracellular value.
    Figure Legend Snippet: Effect of the Ca 2+ mobilizer thapsigargin on the intracellular Ca 2+ concentration of the quiescent cells. Sperm were loaded with calcium green1-AM as described in Materials and Methods and suspended in NoCaFPS (closed circle) or FPS containing 100 nM Ca 2+ (open diamond) or 1,000 nM Ca 2+ (open circle), and 50 μM thapsigargin and fluorescence from the cells was monitored for 10 min. Control cells were suspended in FPS containing 100 nM Ca 2+ (closed circle), and at t = 10 min, 5 μM A 23187 calcium ionophore was added to the suspension and the fluorescence from the sperm cells was measured. When 5 μM A 23187 was added to the sperm cells containing different calcium concentrations and thapsigargin (arrow), the intracellular calcium concentration reached that of the extracellular value.

    Techniques Used: Concentration Assay, Fluorescence

    32) Product Images from "Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat"

    Article Title: Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat

    Journal:

    doi: 10.1002/glia.20148

    Inhibition of morphine (Morph) and/and Tat 1-72- induced increases in intracellular calcium [Ca 2+ ] i by thapsigargin, dantrolene, and the PI3-kinase inhibitor, LY294002. Cells were incubated with serum free medium or stimulated with 500 nM morphine ±
    Figure Legend Snippet: Inhibition of morphine (Morph) and/and Tat 1-72- induced increases in intracellular calcium [Ca 2+ ] i by thapsigargin, dantrolene, and the PI3-kinase inhibitor, LY294002. Cells were incubated with serum free medium or stimulated with 500 nM morphine ±

    Techniques Used: Inhibition, Incubation

    33) Product Images from "Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation"

    Article Title: Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation

    Journal: The Open Biochemistry Journal

    doi: 10.2174/1874091X00802010091

    Comparison of enzyme activities in isolated SR vesicles and open membrane patches from a single preparation. Enzyme activity was determined by the coupled pyruvate kinase/lactate dehydrogenase assay [ 20 ]. In both columns 1 enzyme activity is shown without further additions, in columns 2 the activity is shown after addition of 12.5 µM of the Ca ionophore A23187. In the case of the SR vesicles a significant increase of the enzyme activity was observed due to the disappearance of the Ca 2+ concentration gradient across the SR membrane. As expected, in the open mem-branes the ionophore had no amplifying effect. In columns 3 the action of the specific inhibitor thapsigargin is demon-strated. The negligible remaining enzyme activity proves that no significant amounts of other ATPase are present in both preparations.
    Figure Legend Snippet: Comparison of enzyme activities in isolated SR vesicles and open membrane patches from a single preparation. Enzyme activity was determined by the coupled pyruvate kinase/lactate dehydrogenase assay [ 20 ]. In both columns 1 enzyme activity is shown without further additions, in columns 2 the activity is shown after addition of 12.5 µM of the Ca ionophore A23187. In the case of the SR vesicles a significant increase of the enzyme activity was observed due to the disappearance of the Ca 2+ concentration gradient across the SR membrane. As expected, in the open mem-branes the ionophore had no amplifying effect. In columns 3 the action of the specific inhibitor thapsigargin is demon-strated. The negligible remaining enzyme activity proves that no significant amounts of other ATPase are present in both preparations.

    Techniques Used: Isolation, Activity Assay, Lactate Dehydrogenase Assay, Concentration Assay

    34) Product Images from "Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration *"

    Article Title: Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.668996

    MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with thapsigargin abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p
    Figure Legend Snippet: MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with thapsigargin abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p

    Techniques Used: Activation Assay, Concentration Assay, Inhibition, Immunocytochemistry, Staining, Labeling, Expressing

    35) Product Images from "The arrestin-domain containing protein AdcA is a response element to stress"

    Article Title: The arrestin-domain containing protein AdcA is a response element to stress

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-91

    Effect of various mutants and addition of 8-Br-cAMP, 8-Br-cGMP and thapsigargin on AdcA phosphorylation. A . AdcA is not phosphorylated in response to 8-Br-cAMP. Vegetative or 4 h-starved cells in KK2 buffer (16.5 mM KH 2 PO 4 , 3.8 mM K 2 HPO 4 , pH 6.2) were treated with 20 mM 8-Br-cAMP or 200 mM sorbitol. B . AdcA is not dependent on DokA. dokA null cells kept in nutritive medium or starved for 4 h in KK2 buffer were treated with 200 mM sorbitol. C . AdcA is partly phosphorylated in response to a combination of 8-Br-cGMP and thapsigargin. Vegetative KAx-3 cells were treated with 20 mM 8-Br-cGMP, 10 μM thapsigargin or a mix of 8-Br-cGMP/thapsigargin. D . The response of AdcA is not affected in sgc / gca double null mutant. The sgc/gca null strain disrupted for the 2 guanylate cyclases sGC and GCA were subjected to 200 mM sorbitol. In all tested conditions, the response of AdcA was followed by Western blot using anti-AdcA antibodies (A, B, C and D) . The phosphorylation of STATc in the same conditions was detected using the 3H7 and CP22 antibodies and was used as a positive control of cell responsiveness (A and C) .
    Figure Legend Snippet: Effect of various mutants and addition of 8-Br-cAMP, 8-Br-cGMP and thapsigargin on AdcA phosphorylation. A . AdcA is not phosphorylated in response to 8-Br-cAMP. Vegetative or 4 h-starved cells in KK2 buffer (16.5 mM KH 2 PO 4 , 3.8 mM K 2 HPO 4 , pH 6.2) were treated with 20 mM 8-Br-cAMP or 200 mM sorbitol. B . AdcA is not dependent on DokA. dokA null cells kept in nutritive medium or starved for 4 h in KK2 buffer were treated with 200 mM sorbitol. C . AdcA is partly phosphorylated in response to a combination of 8-Br-cGMP and thapsigargin. Vegetative KAx-3 cells were treated with 20 mM 8-Br-cGMP, 10 μM thapsigargin or a mix of 8-Br-cGMP/thapsigargin. D . The response of AdcA is not affected in sgc / gca double null mutant. The sgc/gca null strain disrupted for the 2 guanylate cyclases sGC and GCA were subjected to 200 mM sorbitol. In all tested conditions, the response of AdcA was followed by Western blot using anti-AdcA antibodies (A, B, C and D) . The phosphorylation of STATc in the same conditions was detected using the 3H7 and CP22 antibodies and was used as a positive control of cell responsiveness (A and C) .

    Techniques Used: Mutagenesis, Western Blot, Positive Control

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    Alomone Labs thapsigargin
    Both extracellular and intracellular Ca 2+ stores contribute to leukotoxin-induced [Ca 2+ ] i changes in cerebellar neurones. A. Averaged traces of four recordings obtained at different extracellular Ca 2+ concentrations ([Ca 2+ ] e ) showing that, in a Ca 2+ -free medium, neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca 2+ ] e (3 μM), an increase in free [Ca 2+ ] i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 μM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 μM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 μM <t>thapsigargin;</t> two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 μM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. C. Lysosomal destruction by 0.2 μM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca 2+ ] i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke.
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    Both extracellular and intracellular Ca 2+ stores contribute to leukotoxin-induced [Ca 2+ ] i changes in cerebellar neurones. A. Averaged traces of four recordings obtained at different extracellular Ca 2+ concentrations ([Ca 2+ ] e ) showing that, in a Ca 2+ -free medium, neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca 2+ ] e (3 μM), an increase in free [Ca 2+ ] i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 μM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 μM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 μM thapsigargin; two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 μM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. C. Lysosomal destruction by 0.2 μM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca 2+ ] i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke.

    Journal: Cellular Microbiology

    Article Title: Staphylococcal leukotoxins trigger free intracellular Ca2+ rise in neurones, signalling through acidic stores and activation of store-operated channels

    doi: 10.1111/cmi.12069

    Figure Lengend Snippet: Both extracellular and intracellular Ca 2+ stores contribute to leukotoxin-induced [Ca 2+ ] i changes in cerebellar neurones. A. Averaged traces of four recordings obtained at different extracellular Ca 2+ concentrations ([Ca 2+ ] e ) showing that, in a Ca 2+ -free medium, neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca 2+ ] e (3 μM), an increase in free [Ca 2+ ] i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 μM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 μM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 μM thapsigargin; two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 μM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. C. Lysosomal destruction by 0.2 μM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca 2+ ] i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke.

    Article Snippet: The voltage-gated channel blockers Tetrodotoxin, ω-Conotoxin GVI-A, ω-Agatoxin TK as well as ryanodine and thapsigargin were from Alomone Labs (Israel).

    Techniques:

    Ca 2+ store depletion does not affect TRPC channel activation by kisspeptin. A–C, Representative recordings showing the kisspeptin (Kp-10)-induced inward current after a GnRH neuron had been exposed to thapsigargin (Tg, 1μM) for 10 minutes

    Journal: Endocrinology

    Article Title: Kisspeptin Activation of TRPC4 Channels in Female GnRH Neurons Requires PIP2 Depletion and cSrc Kinase Activation

    doi: 10.1210/en.2013-1180

    Figure Lengend Snippet: Ca 2+ store depletion does not affect TRPC channel activation by kisspeptin. A–C, Representative recordings showing the kisspeptin (Kp-10)-induced inward current after a GnRH neuron had been exposed to thapsigargin (Tg, 1μM) for 10 minutes

    Article Snippet: The following chemicals or drugs (see ) were used: kisspeptin-10 (mouse Kiss-1 [110-119]-NH2 ; Phoenix Pharmaceuticals, Belmont, California); TTX and thapsigargin (Alomone Laboratories, Jerusalem, Israel); phorbol 12,13-dibutyrate (PDBu), wortmannin, genistein, PP2 (3,4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4- d ]pyrimidin-4-amine) (Sigma-Aldrich, St Louis, Missouri); calphostin C, bisindolylmaleimide-I (BIS-I), PP3 (1-phenyl-1H-pyrazolo[3,4- d ]pyrimidin-4-amine), amlodipine, AA, and U0126 (Tocris, Ellisville, Missouri); myo-IP3 and OAG (Avanti Polar Lipids, Alabaster, Alabama); and Dioctanoylglycerol-PIP2 (DiC8-PIP2 ) (Echelon Biosciences, Salt Lake City, Utah).

    Techniques: Activation Assay

    Germ cells are viable in the SST and display spontaneous Ca 2+ oscillations. Ca 2+ recordings from germ cells in the SST corresponding to the addition of 10 μM thapsigargin ( A ) or 120 mM KCl ( B ). C ) Fluorescence traces obtained from the four cells

    Journal: Biology of Reproduction

    Article Title: Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters 1

    doi: 10.1095/biolreprod.112.100255

    Figure Lengend Snippet: Germ cells are viable in the SST and display spontaneous Ca 2+ oscillations. Ca 2+ recordings from germ cells in the SST corresponding to the addition of 10 μM thapsigargin ( A ) or 120 mM KCl ( B ). C ) Fluorescence traces obtained from the four cells

    Article Snippet: Niquel (Ni2+ ), mibefradil, T (Sigma-Aldrich), and thapsigargin (Alomone) were applied on the perfusion system during recordings, and 18α-glycyrrhetinic acid (Sigma-Aldrich) was incubated during 10 min between control and experimental condition.

    Techniques: Fluorescence

    Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p

    Journal: The Journal of Neuroscience

    Article Title: Calcium-Activated Sustained Firing Responses Distinguish Accessory from Main Olfactory Bulb Mitral Cells

    doi: 10.1523/JNEUROSCI.4397-11.2012

    Figure Lengend Snippet: Analysis of I can induction after a spike train in AOB mitral cells. A , The hybrid-clamp protocol. A burst of spikes was elicited in current-clamp mode by a depolarizing current pulse train (duration, 4 s; rate, 20 Hz; amplitude, 300 pA; width, 10 ms), followed by a switch to voltage-clamp mode. The hybrid-clamp modes are indicated in the bar above. The dashed line gives the baseline current. The stimulus train induced a transient outward current followed by a slow prolonged inward current of ∼10 pA. B , The protocol for measuring the reversal potential of the prolonged current. Several voltage ramps were given before and after the spike train (marked by a green bar). C , I–V curves before and after the pulse train, using the ramps color coded in B . The conductance was calculated from the change in slope between the curves, and the reversal potential was calculated from the intersection of their extrapolated linear fits. D , The averaged current after the pulse train (green bar) to AOB mitral cells in the control condition (dotted line, 9 cells) and in slices incubated with blockers of N- and R-type VACCs (continuous line, 9 cells). E , The inverse of the charge transferred in the prolonged current from 2 to 34 s after spike train (left) in control conditions (green, 8 cells) and after intracellular calcium stores depletion by thapsigargin (magenta, 3 cells) or after a DHPG puff (right) in the same conditions (control, 10 cells; thapsigargin, 6 cells). Bars and error bars represent mean ± SEM; Mann–Whitney U test, * p

    Article Snippet: For depletion of intracellular calcium stores, thapsigargin (2 μ m ; Alomone Labs) was added to the bath solution. ( RS )-3,5-Dihydroxyphenylglycine (DHPG) (Sigma) was delivered locally by pressure pulses(2-s-long) via a glass pipette containing 200 μ m DHPG dissolved in physiological solution.

    Techniques: Mass Spectrometry, Incubation, MANN-WHITNEY