thapsigargin  (Alomone Labs)


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    Structured Review

    Alomone Labs thapsigargin
    SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with <t>thapsigargin</t> (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p
    Thapsigargin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 3 article reviews
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    thapsigargin - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering"

    Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

    Journal: bioRxiv

    doi: 10.1101/2022.02.21.481295

    SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with thapsigargin (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p
    Figure Legend Snippet: SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with thapsigargin (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p

    Techniques Used: Activation Assay, Transfection, Live Cell Imaging, Confocal Microscopy, Positive Control, Transmission Electron Microscopy

    2) 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

    3) Product Images from "SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System"

    Article Title: SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System

    Journal: Stem Cell Reviews and Reports

    doi: 10.1007/s12015-021-10179-x

    iSOX9-astrocytes show comparable calcium responses as fHA. Calcium responses (plotted as F340/F380 ratio) measured in Fura-2-loaded iSOX9-astrocytes and fHA in the presence of extracellular Ca 2+ -chelating agent BAPTA (3 mM), ensuring only Ca 2+ release from internal Ca 2+ stores is measured. a After stimulating fHA and iSOX9-astrocytes with ATP, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). b After stimulating fHA and iSOX9-astrocytes with Ach, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). c iSOX9-astrocytes and fHA show a similar calcium response after stimulation with GPN, FCCP and ionomycin measured by Fura2 fluorescence emission. A significantly higher calcium response is observed in fHA compared to iSOX9-astrocytes after thapsigargin stimulation (**p
    Figure Legend Snippet: iSOX9-astrocytes show comparable calcium responses as fHA. Calcium responses (plotted as F340/F380 ratio) measured in Fura-2-loaded iSOX9-astrocytes and fHA in the presence of extracellular Ca 2+ -chelating agent BAPTA (3 mM), ensuring only Ca 2+ release from internal Ca 2+ stores is measured. a After stimulating fHA and iSOX9-astrocytes with ATP, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). b After stimulating fHA and iSOX9-astrocytes with Ach, they show a similar calcium response measured by Fura2 fluorescence emission (N = 6 independent differentiations). c iSOX9-astrocytes and fHA show a similar calcium response after stimulation with GPN, FCCP and ionomycin measured by Fura2 fluorescence emission. A significantly higher calcium response is observed in fHA compared to iSOX9-astrocytes after thapsigargin stimulation (**p

    Techniques Used: Fluorescence

    4) 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

    5) 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:

    6) 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:

    7) 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:

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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:

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    Alomone Labs thapsigargin
    SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with <t>thapsigargin</t> (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p
    Thapsigargin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with thapsigargin (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p

    Journal: bioRxiv

    Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

    doi: 10.1101/2022.02.21.481295

    Figure Lengend Snippet: SFK activation does not enhance mitochondrial fusion, mitophagy, or ER stress signaling. A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also Fig. 3 ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with thapsigargin (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p

    Article Snippet: All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) except for: 2- mercaptoethanol (Bio-Rad, Hercules, CA); Proteinase K and dithiothreitol (DTT) (Gentrox, Shrewsberry, MA); Mito Tracker Deep Red and tetramethylrhodamine, ethyl ester (TMRE) (Thermo Fisher Scientific, , Waltham, MA); PP2 (Cayman Chemical, Ann Arbor, Michigan); Torin 1 (LC Laboratories, Woburn, MA); and thapsigargin (Alomone Labs, Jerusalem, Israel).

    Techniques: Activation Assay, Transfection, Live Cell Imaging, Confocal Microscopy, Positive Control, Transmission Electron Microscopy