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  • 99
    Thermo Fisher thapsigargin
    Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with <t>BODIPY</t> FL <t>thapsigargin</t> (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P
    Thapsigargin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore thapsigargin
    Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with <t>BODIPY</t> FL <t>thapsigargin</t> (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P
    Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris thapsigargin
    Cytosolic Ca 2+ triggers cell proliferation and EE differentiation through different mechanisms. a,b. Increase of cytosolic Ca 2+ by channelrhodopsin (ChR) in Dl + and Piezo + EP cells. Dl + , Piezo + , and EE cell numbers are quantified. Number of areas quantified: n=28 (Dark, Dl-Gal4 ), n=30 (Light+ATR, Dl-Gal4 ), n=30 (Dark, Piezo-Gal4 ), n=31 (Light+ATR, Piezo-Gal4 ). c,d. Midguts of <t>Thapsigargin</t> (Thap) and Trametinib (Tram) treated flies. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar, 50 μm.
    Thapsigargin, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem thapsigargin
    Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cell lines are more sensitive to <t>thapsigargin</t> treatment than Cb Cln3 +/+ cells. A, workflow for hit identification and categorization of activities for our primary screening data is shown. B, structure of thapsigargin,
    Thapsigargin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs thapsigargin
    Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM <t>thapsigargin</t> 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
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    Cayman Chemical thapsigargin
    The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM <t>thapsigargin</t> (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p
    Thapsigargin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam thapsigargin
    The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM <t>thapsigargin</t> (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p
    Thapsigargin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM thapsigargin
    Identification of PERK- and eIF2α-binding region. (A) Schematic representations of each TBL2 mutant. (B) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM <t>thapsigargin</t> for 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis. (C) Schematic representations of each TBL2 mutant. (D) HT1080 cells were transiently co-transfected with pTBL2 mutant (FLAG, red) and pTBL2 WT (V5-tag, green) plasmids. After 24 hours, cells were fixed and then analyzed by immunofluorescence using confocal microscope. (E) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 0.5 or 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis.
    Thapsigargin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology thapsigargin
    BA induces an increase in ATF4 transcription in DU-145 cells. Ten micromoles of BA induced a significant increase in ATF4 mRNA 1 ( p = 0.031, n = 4) and 2 h ( p = 0.005, n = 4) post-treatment. One micromole of <t>thapsigargin</t> (Tg) and DMSO vehicle (DM) was used as a positive control and significantly induced ATF4 mRNA, p
    Thapsigargin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LC Laboratories thapsigargin
    <t>Thapsigargin-dependent</t> Ca 2+ entry in S2 cells. Fluo-4 fluorescence changes were monitored using a FLIPR 384 . After 20 s of recording, the 384-well pipette-tip head was lowered into the solution creating an offset artifact in the recording. This offset artifact is unrelated to a cellular response and is dependent on the fluid volume in each well at the start of the experiment and the extent of tip penetration into the solution. 10 s after lowering the pipette-tip head, either thapsigargin (TG, 1 μM final, circles) or DMSO (triangles) was injected (arrow). CaCl 2 was then added to achieve a final concentration of 1.8 mM. Traces were zeroed at time 0, and each data point represents the mean (±SEM) of 192 replicates.
    Thapsigargin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc thapsigargin
    <t>Thapsigargin</t> could not mimic the effects of tunicamycin on MDR GC cells. a Concentration-survival curves of GC cells treated with Tg or Tu (wide dose range) for 48 h. Tg, thapsigargin. b Expressions of UPR-related proteins in GC cells after Tg treatment (2 μg/ml) for 48 h determined by WB. All proteins were normalized to β-actin. c The effects of Tg on the chemosensitivity of GC cells after treatment for 48 h. ns, non-significant; * P
    Thapsigargin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co thapsigargin
    Synergism between ER stress and other stimuli. a FLS ( n = 4) were left untreated or stimulated with 1 μg/ml LPS or 1 ng/ml IL-1β in the presence or absence of 10 nM TG. Expression of IL6 and IL8 was measured by qPCR 4 h or 8 h after stimulation. b , c FLS ( n = 3, n = 2 for TG alone) were stimulated with 10 nM TG or 1 μg/ml LPS in the presence or absence of 10 nM TG for 8 h. Expression of 84 inflammatory genes was monitored by qPCR-based array. Heat map ( b ) depicts per-gene z-scores of log-scaled expression values relative to GAPDH, and bar graph ( c ) presents fold changes relative to unstimulated cells for 30 genes with the highest overall level of regulation (mean across all conditions). ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, IL Interleukin, LPS Lipopolysaccharide, qPCR Quantitative polymerase chain reaction, TG <t>Thapsigargin</t>
    Thapsigargin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH thapsigargin
    SDF-1 and the GLP-1 receptor agonist EXD4 act additively to preserve and enhance beta cell mass. INS-1 cells were cultured in 96 well plates in the presence of 2 nmol/l EXD4 and/or 10 nmol/l SDF-1 on a background of ( a ) serum deprivation or ( b ) serum repletion (0.8%). EXD4 and SDF-1 were replenished every 2 days. Cell viability was measured 6 days after treatment by ATPlite assay. INS-1 cells treated with ( c ) <t>thapsigargin</t> or ( d ) cytokines were incubated with vehicle or 10 nmol/l SDF-1, 10 nmol/l EXD4 or SDF-1+EXD4 for 6 days and their dry weight was then measured. Data are expressed as mass relative to the mass for vehicle treatment. e INS-1 cells were cultured in 96 well plates in the presence of 10 nmol/l SDF-1 with or without AMD3100 (25 μmol/l) on a background of nutrient deprivation by serum withdrawal (no serum). f INS-1 cells were cultured in 96 well plates in the presence of increasing doses of SDF-1 on a background of normal (5 mmol/l) or high glucose concentration (25 mmol/l). Reagents were added at day 0 and day 2, and 4 days after treatment, cell viability was measured using the ATPlite assay. g Dispersed human islets were cultured in 96 well plates (~100 islets/well) in the presence of 2 nmol/l EXD4, and/or 10 nmol/l SDF-1 on a background of cytokines (IL-1β 50 ng/ml, TNFα 10 ng/ml, IFNγ 50 ng/ml). Cell viability was measured 3 days after treatment by ATPlite assay. The viable beta cell number was enhanced additively by EXD4 and SDF-1. Therefore the combination of GLP-1 and SDF-1 increased human islet cell viability against cytokine-stress-induced cell death. h For the insulin secretion assay, INS-1 cells were plated in 96 well plates. SDF-1 (2 nmol/l) and AMD3100 were added at day 0, and the insulin concentration of culture medium was measured at day 6. Statistical significance is depicted as * p
    Thapsigargin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore reagents thapsigargin
    Flx affects osteoclastogenesis in a 5HTT-independent manner ( a ) Quantification of TRAP activity (left) and gene expression in primary osteoclast cultures (OCs) derived from 5HTT-deficient ( Slc6a4 −/− ) mice. Ctsk, Cathepsin K. Clcn7, Chloride channel 7. Acp5 encodes TRAP. ( b ) Representative images ( n = 4 images/mouse) of vertebrae (left) from Slc6a4 −/− females treated for 3 w (veh n = 8, Flx n = 6). Quantification of BV/TV is indicated below each image. Scale bars, 200 μm. Bone histomorphometry of these mice is also indicated (middle and right). ( c , d ) Gene expression in WT OCs ( c ) and Slc6a4 −/− OCs ( d ). Mitf encodes microphthalmia-associated transcription factor; Undiff., undifferentiated OCs. ( e ) Expression of Nfatc1 in long bones of WT females treated for 3 w. ( f ) Western blot (left) and quantification of band intensities reported to veh (right) of indicated proteins in RAW264.7 osteoclasts treated with Flx or veh ( n = 1 technical replicate of the number of biological replicates indicated in the bar graph (left)). ( g ) Representative curves ( n = 13) of Fura-2 ratio (340/380) in individual RAW264.7 osteoclasts recorded in veh medium and after addition of Flx (left), Fura-2 ratio levels after Flx addition relative to veh (AUC, area under the curve ( n = 13) (middle), and proportion of OCs and MNCs responding to Flx (right). <t>Thap,</t> <t>Thapsigargin.</t> ( h ) Gene expression in RAW264.7 osteoclasts treated for 24 h. ( i ) Gene expression in Creb fl/fl:virus-CRE OCs treated for 6 days (6 d) (left), and the percentage of resorbed area in a pit resorption assay of WT OCs treated for 24 h with W5, KG-501 and Flx (right). ( j ) Gene expression in spleen of WT or Slc6a4 −/− females treated for 3 weeks. Rorc encodes RORγt. Values are mean ± SEM compared to veh (*) or undiff. OCs ( $ ) */ $ P ≤ 0.05, ** P ≤ 0.01, ***/ $$$ P ≤ 0.001, ****/ $$$$ P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test ( a , i ), one-way ANOVA followed by Turkey’s ( c , d , h ) or Student’s test (all other panels).
    Reagents Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore 2 4 6 trihydroxyacetophenone monohydrate thap
    Flx affects osteoclastogenesis in a 5HTT-independent manner ( a ) Quantification of TRAP activity (left) and gene expression in primary osteoclast cultures (OCs) derived from 5HTT-deficient ( Slc6a4 −/− ) mice. Ctsk, Cathepsin K. Clcn7, Chloride channel 7. Acp5 encodes TRAP. ( b ) Representative images ( n = 4 images/mouse) of vertebrae (left) from Slc6a4 −/− females treated for 3 w (veh n = 8, Flx n = 6). Quantification of BV/TV is indicated below each image. Scale bars, 200 μm. Bone histomorphometry of these mice is also indicated (middle and right). ( c , d ) Gene expression in WT OCs ( c ) and Slc6a4 −/− OCs ( d ). Mitf encodes microphthalmia-associated transcription factor; Undiff., undifferentiated OCs. ( e ) Expression of Nfatc1 in long bones of WT females treated for 3 w. ( f ) Western blot (left) and quantification of band intensities reported to veh (right) of indicated proteins in RAW264.7 osteoclasts treated with Flx or veh ( n = 1 technical replicate of the number of biological replicates indicated in the bar graph (left)). ( g ) Representative curves ( n = 13) of Fura-2 ratio (340/380) in individual RAW264.7 osteoclasts recorded in veh medium and after addition of Flx (left), Fura-2 ratio levels after Flx addition relative to veh (AUC, area under the curve ( n = 13) (middle), and proportion of OCs and MNCs responding to Flx (right). <t>Thap,</t> <t>Thapsigargin.</t> ( h ) Gene expression in RAW264.7 osteoclasts treated for 24 h. ( i ) Gene expression in Creb fl/fl:virus-CRE OCs treated for 6 days (6 d) (left), and the percentage of resorbed area in a pit resorption assay of WT OCs treated for 24 h with W5, KG-501 and Flx (right). ( j ) Gene expression in spleen of WT or Slc6a4 −/− females treated for 3 weeks. Rorc encodes RORγt. Values are mean ± SEM compared to veh (*) or undiff. OCs ( $ ) */ $ P ≤ 0.05, ** P ≤ 0.01, ***/ $$$ P ≤ 0.001, ****/ $$$$ P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test ( a , i ), one-way ANOVA followed by Turkey’s ( c , d , h ) or Student’s test (all other panels).
    2 4 6 Trihydroxyacetophenone Monohydrate Thap, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM thapsigargin tg
    A CK2 inhibitor inhibited ER stress-induced GRP78 expression. 4,5,6,7-tetrabromobenzotriazole (TBB) inhibited GRP78 expression at the mRNA and protein levels. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and then treated with tunicamycin (Tm: 0.01 µg/mL) or <t>thapsigargin</t> (Tg: 0.01 µM) for 6 h. RT-PCR was performed using specific primers for each mRNA. n = 4/group * p
    Thapsigargin Tg, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA thapsigargin
    NAADP-elicited Ca 2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice ( n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice ( n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM <t>thapsigargin</t> ( n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca 2+ responses to NAADP (100 nM), IP 3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 ( n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.
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    Valiant thapsigargin
    Expression of bbc3/PUMA may be sufficient and required for ER stress–induced apoptosis in human cells. (A) Transient transfection of SH-SY5Y neuroblastoma cells with bbc3/PUMA leads to induction of apoptosis. Cells were cotransfected with expression plasmids encoding enhanced GFP (pEGFP) and either a hemagglutinin-tagged (HA) version of bbc3/PUMA (pHA-PUMA) or empty vector (pcDNA3.1). 12 h after transfection, cells were fixed and stained with Hoechst 33258. Arrowheads point to transfected cells with the apoptotic phenotype. Similar results were obtained in a separate experiment. (B) Quantitative FACS ® analysis of PI uptake of EGFP-positive SH-SY5Y cells transiently cotransfected with expression vectors encoding hemagglutinin-tagged (HA) versions of Bbc3/PUMA, Bbc3/PUMA-ΔBH3, or empty vector (pcDNA3.1). Data were normalized to EGFP-only transfected controls and represent 10,000 events each. For evaluation of comparable transfection rates, whole-cell extracts of cells harvested 16 h after transfection were analyzed by Western blotting. Blots were probed with an anti-hemagglutinin or anti- α-tubulin antibody as loading control. (C) Protein levels of BiP, Bbc3/PUMA, and α-tubulin in human HCT116 control and Bbc3/PUMA-deficient cells treated with 1 μM <t>thapsigargin</t> or vehicle for the indicated time points. (D) FACS ® analysis of sub-G1 cells of human HCT116 control and Bbc3/PUMA-deficient cells exposed to 1 μM thapsigargin (TH) or vehicle for 36 h. (E) Quantitative FACS ® analysis of thapsigargin- and STS (3 μM)-induced apoptosis. Data from 10,000 events each are means ± SEM from n = 3 separate experiments per time point.
    Thapsigargin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with BODIPY FL thapsigargin (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: TNFα decreases mitochondrial movement in human airway smooth muscle

    doi: 10.1152/ajplung.00538.2016

    Figure Lengend Snippet: Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with BODIPY FL thapsigargin (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P

    Article Snippet: For simultaneous visualization of SR and mitochondria, human ASM cells were loaded with 1 µM BODIPY FL thapsigargin (endoplasmic reticulum Ca2+ pumps; Invitrogen; 503 excitation/512 nm emission) for 30 min and then with 300 nM MitoTracker Red CMXRos for 5 min.

    Techniques: Fluorescence

    Cytosolic Ca 2+ triggers cell proliferation and EE differentiation through different mechanisms. a,b. Increase of cytosolic Ca 2+ by channelrhodopsin (ChR) in Dl + and Piezo + EP cells. Dl + , Piezo + , and EE cell numbers are quantified. Number of areas quantified: n=28 (Dark, Dl-Gal4 ), n=30 (Light+ATR, Dl-Gal4 ), n=30 (Dark, Piezo-Gal4 ), n=31 (Light+ATR, Piezo-Gal4 ). c,d. Midguts of Thapsigargin (Thap) and Trametinib (Tram) treated flies. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar, 50 μm.

    Journal: Nature

    Article Title: Mechanical regulation of stem cell differentiation through stretch-activated Piezo channel

    doi: 10.1038/nature25744

    Figure Lengend Snippet: Cytosolic Ca 2+ triggers cell proliferation and EE differentiation through different mechanisms. a,b. Increase of cytosolic Ca 2+ by channelrhodopsin (ChR) in Dl + and Piezo + EP cells. Dl + , Piezo + , and EE cell numbers are quantified. Number of areas quantified: n=28 (Dark, Dl-Gal4 ), n=30 (Light+ATR, Dl-Gal4 ), n=30 (Dark, Piezo-Gal4 ), n=31 (Light+ATR, Piezo-Gal4 ). c,d. Midguts of Thapsigargin (Thap) and Trametinib (Tram) treated flies. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar, 50 μm.

    Article Snippet: 4μM DAPT (Sigma, D5942), 10μg/ml Bleomycin (Calbiochem #203408), 0.5μM Thapsigargin (Tocris, 1138), and 10μM Trametinib (Selleckchem, S2673) were used for chemical treatment.

    Techniques: CTL Assay, Two Tailed Test

    Cytosolic Ca 2+ triggers ISC proliferation and EP differentiation into EEs. a. Image of chamber used for optogenetic activation of ChR. b,c. Flies expressing GFP only in Dl + stem cells or Piezo + EP (EE precursor) cells were treated under either dark or light + ATR condition for two weeks like the flies expressing ChR. No significant phenotype was induced by the treatment alone. Number of midgut areas quantified: n=29 (Dl, Dark), n=33 (Dl, light+ATR), n=31 (Piezo, Dark), n=34 (Piezo, light+ATR). Representative results from two independent replicates are shown. d. Mitosis quantification of midgut from indicated genotype/condition. Activating ChR in Dl + cells significantly promotes stem cell proliferation. Only a mild increase of mitosis was detected in ChR active Piezo + EP cells, suggesting that the primary effect of Ca 2+ in EP cells is to promote differentiation. Data are collected from 30 guts ( Dl > ChR ); 30 guts ( Piezo > ChR ); 29 guts ( Dl ); guts ( Piezo ) from two independent replicates. pH3 + cell number is quantified from the whole midgut. e,f. Activation of CsChrimson in Dl + stem cells with both Stim and InsP3R area from 29 regions (dark) and 31 regions (light + ATR) from two independent replicates. g. Overexpression of Piezo in esg + cells increases MAPK pathway activity. Phosphorylation of extracellular signal-regulated kinase (dpErk) is significantly increased in Piezo -overexpressing cells. Representative images from two independent experiments are shown. h,i. Knocking down Ras significantly reduces stem cell proliferation caused by Piezo overexpression, but does not block Piezo triggered EE differentiation. Flies were kept at 32°C for 4–5 days before analysis. esg + and EE cell number were quantified from n=29 (control) and n=30 ( Piezo GFP ) midguts areas from two independent experiments. “Newborn” EEs, that are positive for both esg and Pros, are indicated by arrowheads. j,k. Knocking-down Yorkie using Yki RNAi completely blocks stem cell proliferation but not the increase of EE cells induced by either Piezo overexpression or Serca knock-down. In addition, knocking-down Serca together with Yorkie also significantly reduced stem cell number, suggesting a depletion of stem cells caused by constant EE differentiation. Cell numbers were quantified within 30 midgut areas for each genotype. l. Midguts from flies fed on control (5% sucrose), Thap (5% sucrose + 0.5μM Thapsigargin), Thap+Tram (5% sucrose + 0.5μM Thapsigargin + 10μM Trametinib), and Tram (5% sucrose + 5μM Trametinib) for 4 days. Representative images from 3 independent experiments are shown. The increase of cytosolic Ca 2+ by Thap promotes stem cell proliferation, EP (enteroendocrine precursor/Piezo + cell) production, and EE differentiation. Newborn EEs, which are positive for esg, Piezo and Pros, are indicated by white arrowheads. m . Data are collected from Quantification of mitotic cells from n=15 (control), n=16 (Thap), n=17 (Thap + Tram), and n=16 (Tram) midguts. Thap treatment triggers a significant increase in mitosis, which is largely reduced by the mitogen-activated protein kinase (MAP kinase) inhibitor Tram. n. Percentage of Piezo + cells within esg + cell population. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). o. Representative Ca 2+ images of live midgut from control, Thap, and Thap+Tram treated flies. Similar results are collected from 4 independent guts for each condition. p,q. Thap treatment caused a reduction of oscillation frequency but an increase of average GCaMP/RFP ratio (G/R ratio). The increase of cytosolic Ca 2+ by Thap is not affected by MAPK inhibition. Data are collected from 29 Cells from 3 independent guts for each condition. All data are expressed as mean + s.e.m. values (shown in red). P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: 50 μm.

    Journal: Nature

    Article Title: Mechanical regulation of stem cell differentiation through stretch-activated Piezo channel

    doi: 10.1038/nature25744

    Figure Lengend Snippet: Cytosolic Ca 2+ triggers ISC proliferation and EP differentiation into EEs. a. Image of chamber used for optogenetic activation of ChR. b,c. Flies expressing GFP only in Dl + stem cells or Piezo + EP (EE precursor) cells were treated under either dark or light + ATR condition for two weeks like the flies expressing ChR. No significant phenotype was induced by the treatment alone. Number of midgut areas quantified: n=29 (Dl, Dark), n=33 (Dl, light+ATR), n=31 (Piezo, Dark), n=34 (Piezo, light+ATR). Representative results from two independent replicates are shown. d. Mitosis quantification of midgut from indicated genotype/condition. Activating ChR in Dl + cells significantly promotes stem cell proliferation. Only a mild increase of mitosis was detected in ChR active Piezo + EP cells, suggesting that the primary effect of Ca 2+ in EP cells is to promote differentiation. Data are collected from 30 guts ( Dl > ChR ); 30 guts ( Piezo > ChR ); 29 guts ( Dl ); guts ( Piezo ) from two independent replicates. pH3 + cell number is quantified from the whole midgut. e,f. Activation of CsChrimson in Dl + stem cells with both Stim and InsP3R area from 29 regions (dark) and 31 regions (light + ATR) from two independent replicates. g. Overexpression of Piezo in esg + cells increases MAPK pathway activity. Phosphorylation of extracellular signal-regulated kinase (dpErk) is significantly increased in Piezo -overexpressing cells. Representative images from two independent experiments are shown. h,i. Knocking down Ras significantly reduces stem cell proliferation caused by Piezo overexpression, but does not block Piezo triggered EE differentiation. Flies were kept at 32°C for 4–5 days before analysis. esg + and EE cell number were quantified from n=29 (control) and n=30 ( Piezo GFP ) midguts areas from two independent experiments. “Newborn” EEs, that are positive for both esg and Pros, are indicated by arrowheads. j,k. Knocking-down Yorkie using Yki RNAi completely blocks stem cell proliferation but not the increase of EE cells induced by either Piezo overexpression or Serca knock-down. In addition, knocking-down Serca together with Yorkie also significantly reduced stem cell number, suggesting a depletion of stem cells caused by constant EE differentiation. Cell numbers were quantified within 30 midgut areas for each genotype. l. Midguts from flies fed on control (5% sucrose), Thap (5% sucrose + 0.5μM Thapsigargin), Thap+Tram (5% sucrose + 0.5μM Thapsigargin + 10μM Trametinib), and Tram (5% sucrose + 5μM Trametinib) for 4 days. Representative images from 3 independent experiments are shown. The increase of cytosolic Ca 2+ by Thap promotes stem cell proliferation, EP (enteroendocrine precursor/Piezo + cell) production, and EE differentiation. Newborn EEs, which are positive for esg, Piezo and Pros, are indicated by white arrowheads. m . Data are collected from Quantification of mitotic cells from n=15 (control), n=16 (Thap), n=17 (Thap + Tram), and n=16 (Tram) midguts. Thap treatment triggers a significant increase in mitosis, which is largely reduced by the mitogen-activated protein kinase (MAP kinase) inhibitor Tram. n. Percentage of Piezo + cells within esg + cell population. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). o. Representative Ca 2+ images of live midgut from control, Thap, and Thap+Tram treated flies. Similar results are collected from 4 independent guts for each condition. p,q. Thap treatment caused a reduction of oscillation frequency but an increase of average GCaMP/RFP ratio (G/R ratio). The increase of cytosolic Ca 2+ by Thap is not affected by MAPK inhibition. Data are collected from 29 Cells from 3 independent guts for each condition. All data are expressed as mean + s.e.m. values (shown in red). P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: 50 μm.

    Article Snippet: 4μM DAPT (Sigma, D5942), 10μg/ml Bleomycin (Calbiochem #203408), 0.5μM Thapsigargin (Tocris, 1138), and 10μM Trametinib (Selleckchem, S2673) were used for chemical treatment.

    Techniques: Activation Assay, Expressing, Over Expression, Activity Assay, Blocking Assay, CTL Assay, Inhibition, Two Tailed Test

    ER stress induces the production of CXCL10 and CCL2 in photoreceptor cells Photoreceptor cell line 661W was treated with 0.1% DMSO (veh) or 0.01 μM thapsigargin (TG). (A–G) Cells were collected for mRNA extraction at indicated time points. The mRNA levels of ER stress-related genes (GRP78, ATF4, CHOP, XBP1s and ATF6), CXCL10 and CCL2 were analyze by qPCR. *p

    Journal: Experimental eye research

    Article Title: PERK and XBP1 differentially regulate CXCL10 and CCL2 production

    doi: 10.1016/j.exer.2017.01.002

    Figure Lengend Snippet: ER stress induces the production of CXCL10 and CCL2 in photoreceptor cells Photoreceptor cell line 661W was treated with 0.1% DMSO (veh) or 0.01 μM thapsigargin (TG). (A–G) Cells were collected for mRNA extraction at indicated time points. The mRNA levels of ER stress-related genes (GRP78, ATF4, CHOP, XBP1s and ATF6), CXCL10 and CCL2 were analyze by qPCR. *p

    Article Snippet: All inhibitors were performed 30 minutes before thapsigargin (TG) (Tocris Bioscience, Minneapolis, MN) treatment.

    Techniques: Real-time Polymerase Chain Reaction

    Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cell lines are more sensitive to thapsigargin treatment than Cb Cln3 +/+ cells. A, workflow for hit identification and categorization of activities for our primary screening data is shown. B, structure of thapsigargin,

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cell lines are more sensitive to thapsigargin treatment than Cb Cln3 +/+ cells. A, workflow for hit identification and categorization of activities for our primary screening data is shown. B, structure of thapsigargin,

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques:

    Sensitivity of CbCln3Δex7/8/Δex7/8 Cells to Thapsigargin Treatment Is Not Due to a General ER Stress Response, but Is Ca2+ -related

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Sensitivity of CbCln3Δex7/8/Δex7/8 Cells to Thapsigargin Treatment Is Not Due to a General ER Stress Response, but Is Ca2+ -related

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques:

    Thapsigargin and autophagy analysis in human iPSC-derived neural progenitor cells from control and JNCL-affected subjects. A, representative epifluorescence images are shown from control (unaffected) and affected JNCL subject ( CLN3 IVS13/E15 and CLN3 Δ

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Thapsigargin and autophagy analysis in human iPSC-derived neural progenitor cells from control and JNCL-affected subjects. A, representative epifluorescence images are shown from control (unaffected) and affected JNCL subject ( CLN3 IVS13/E15 and CLN3 Δ

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques: Derivative Assay

    Thapsigargin effect is mediated by Ca 2+ . For A, B, E, and F , representative epifluorescence images are shown for wild type (Cb Cln3 +/+ ) and homozygous Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells stably expressing GFP-LC3 ( green ) ( A and E ) or control versus

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Thapsigargin effect is mediated by Ca 2+ . For A, B, E, and F , representative epifluorescence images are shown for wild type (Cb Cln3 +/+ ) and homozygous Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells stably expressing GFP-LC3 ( green ) ( A and E ) or control versus

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques: Stable Transfection, Expressing

    Ca 2+ analysis in response to thapsigargin treatment. A, bar graph depicts Ca 2+ measurements in Cb Cln3 +/+ ( white bars ) and Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells ( black bars ) using Fura-2AM (baseline normalized, Δ F / F 0 ) following low-dose (100

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Ca 2+ analysis in response to thapsigargin treatment. A, bar graph depicts Ca 2+ measurements in Cb Cln3 +/+ ( white bars ) and Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells ( black bars ) using Fura-2AM (baseline normalized, Δ F / F 0 ) following low-dose (100

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques:

    Thapsigargin induces p62/SQSTM1 accumulation in mouse cerebellar cells and human NPCs. A, representative confocal images are shown for p62 immunostaining (anti-p62, red ) of wild type (Cb Cln3 +/+ ) and homozygous Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells

    Journal: The Journal of Biological Chemistry

    Article Title: Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function *

    doi: 10.1074/jbc.M114.621706

    Figure Lengend Snippet: Thapsigargin induces p62/SQSTM1 accumulation in mouse cerebellar cells and human NPCs. A, representative confocal images are shown for p62 immunostaining (anti-p62, red ) of wild type (Cb Cln3 +/+ ) and homozygous Cb Cln3 Δ ex7 / 8 /Δ ex7 / 8 cells

    Article Snippet: The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technologies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126).

    Techniques: Immunostaining

    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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

    Figure Lengend 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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: 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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

    Figure Lengend 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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: 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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

    Figure Lengend 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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: 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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

    Figure Lengend 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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: 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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

    Figure Lengend 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

    Article Snippet: Thapsigargin was from Alomone Labs.

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

    The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM thapsigargin (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p

    Journal: PLoS ONE

    Article Title: Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

    doi: 10.1371/journal.pone.0195780

    Figure Lengend Snippet: The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction. Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM thapsigargin (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p

    Article Snippet: Pharmacological inhibitors nitrendipine, nifedipine, thapsigargin, U0126, mibefradil and Y-27632 were purchased from Cayman Chemicals (Ann Arbor, MI).

    Techniques: Standard Deviation, Incubation

    Identification of PERK- and eIF2α-binding region. (A) Schematic representations of each TBL2 mutant. (B) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis. (C) Schematic representations of each TBL2 mutant. (D) HT1080 cells were transiently co-transfected with pTBL2 mutant (FLAG, red) and pTBL2 WT (V5-tag, green) plasmids. After 24 hours, cells were fixed and then analyzed by immunofluorescence using confocal microscope. (E) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 0.5 or 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis.

    Journal: PLoS ONE

    Article Title: TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0112761

    Figure Lengend Snippet: Identification of PERK- and eIF2α-binding region. (A) Schematic representations of each TBL2 mutant. (B) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis. (C) Schematic representations of each TBL2 mutant. (D) HT1080 cells were transiently co-transfected with pTBL2 mutant (FLAG, red) and pTBL2 WT (V5-tag, green) plasmids. After 24 hours, cells were fixed and then analyzed by immunofluorescence using confocal microscope. (E) 293T cells were transiently transfected with each TBL2 mutant plasmid and then treated with 300 nM thapsigargin for 0.5 or 1 h. After immunoprecipitation, each sample was subjected to immunoblot analysis.

    Article Snippet: Tunicamycin (Nacalai Tesque) and thapsigargin (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in dimethyl sulfoxide.

    Techniques: Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Microscopy

    Effects of TBL2 knockdown on the PERK pathway. (A) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). After 48 h, the cells were treated with 300 nM thapsigargin for 90 min and analyzed by immunoblot anaysis. (B) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). The protein synthesis rate was measured by incorporating [ 35 S]methionine/cysteine. The pulse labeling was carried out during the last 20 min of the 40-min thapsigargin (Tg) treatment (100 or 300 nM). Upper: autoradiography image of SDS-PAGE. Lower panel: TCA precipitation sample was measured using a scintillation counter.

    Journal: PLoS ONE

    Article Title: TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0112761

    Figure Lengend Snippet: Effects of TBL2 knockdown on the PERK pathway. (A) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). After 48 h, the cells were treated with 300 nM thapsigargin for 90 min and analyzed by immunoblot anaysis. (B) 293 cells were transiently transfected with non-silencing siRNA, two TBL2 siRNAs (#1, #2) or PERK siRNAs (#1, #2). The protein synthesis rate was measured by incorporating [ 35 S]methionine/cysteine. The pulse labeling was carried out during the last 20 min of the 40-min thapsigargin (Tg) treatment (100 or 300 nM). Upper: autoradiography image of SDS-PAGE. Lower panel: TCA precipitation sample was measured using a scintillation counter.

    Article Snippet: Tunicamycin (Nacalai Tesque) and thapsigargin (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in dimethyl sulfoxide.

    Techniques: Transfection, Labeling, Autoradiography, SDS Page, TCA Precipitation

    Preferential binding of TBL2 to phospho-PERK. (A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and either pFLAG-PERK, pFLAG-PERK(K621A) or pFLAG-IRE1 and then were treated with 300 nM thapsigargin (Tg) for 2 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg), 4 µg/ml tunicamycin (Tu) or 10 mM 2-deoxyglucose (2DG) for 2 h. Endogenous PERK protein was detected with anti-PERK or anti–phospho-PERK antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with the indicated doses of hydrogen peroxide (H 2 O 2 ) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody. (D) 786-O, 293 and 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg) for 1 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.

    Journal: PLoS ONE

    Article Title: TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0112761

    Figure Lengend Snippet: Preferential binding of TBL2 to phospho-PERK. (A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and either pFLAG-PERK, pFLAG-PERK(K621A) or pFLAG-IRE1 and then were treated with 300 nM thapsigargin (Tg) for 2 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg), 4 µg/ml tunicamycin (Tu) or 10 mM 2-deoxyglucose (2DG) for 2 h. Endogenous PERK protein was detected with anti-PERK or anti–phospho-PERK antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with the indicated doses of hydrogen peroxide (H 2 O 2 ) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody. (D) 786-O, 293 and 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg) for 1 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.

    Article Snippet: Tunicamycin (Nacalai Tesque) and thapsigargin (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in dimethyl sulfoxide.

    Techniques: Binding Assay, Transfection, Immunoprecipitation

    TBL2 interacts with PERK in response to ER stress. (A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and pFLAG-PERK, and then were treated with 300 nM thapsigargin for 1, or 3 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-PERK or together with pTBL2 (non-tag). After immunoprecipitation with anti-FLAG conjugated beads, PERK-bound TBL2 protein was detected with anti-TBL2 antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin for 1 hour, 1 mM DTT for 30 min or with 5 mM histidinol (HisOH) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.

    Journal: PLoS ONE

    Article Title: TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0112761

    Figure Lengend Snippet: TBL2 interacts with PERK in response to ER stress. (A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and pFLAG-PERK, and then were treated with 300 nM thapsigargin for 1, or 3 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-PERK or together with pTBL2 (non-tag). After immunoprecipitation with anti-FLAG conjugated beads, PERK-bound TBL2 protein was detected with anti-TBL2 antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin for 1 hour, 1 mM DTT for 30 min or with 5 mM histidinol (HisOH) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.

    Article Snippet: Tunicamycin (Nacalai Tesque) and thapsigargin (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in dimethyl sulfoxide.

    Techniques: Transfection, Immunoprecipitation

    BA induces an increase in ATF4 transcription in DU-145 cells. Ten micromoles of BA induced a significant increase in ATF4 mRNA 1 ( p = 0.031, n = 4) and 2 h ( p = 0.005, n = 4) post-treatment. One micromole of thapsigargin (Tg) and DMSO vehicle (DM) was used as a positive control and significantly induced ATF4 mRNA, p

    Journal: Biological Trace Element Research

    Article Title: Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

    doi: 10.1007/s12011-016-0824-y

    Figure Lengend Snippet: BA induces an increase in ATF4 transcription in DU-145 cells. Ten micromoles of BA induced a significant increase in ATF4 mRNA 1 ( p = 0.031, n = 4) and 2 h ( p = 0.005, n = 4) post-treatment. One micromole of thapsigargin (Tg) and DMSO vehicle (DM) was used as a positive control and significantly induced ATF4 mRNA, p

    Article Snippet: Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Positive Control

    BA does not activate the IRE1 signaling pathway . a DU-145 cells were treated with 0, 10, 50, 100, or 250 μM BA or 1 μM thapsigargin (Tg) for 24 h. Thapsigargin was used as a positive control. Increasing BA concentrations for 24 h did not cause XBP1 cleavage. b DU-145 cells were treated with 10 μM BA for varying time points or 1 μM thapsigargin (Tg) as a positive control. XBP1 was not cleaved at any time point by BA, but it was by Tg ( p

    Journal: Biological Trace Element Research

    Article Title: Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

    doi: 10.1007/s12011-016-0824-y

    Figure Lengend Snippet: BA does not activate the IRE1 signaling pathway . a DU-145 cells were treated with 0, 10, 50, 100, or 250 μM BA or 1 μM thapsigargin (Tg) for 24 h. Thapsigargin was used as a positive control. Increasing BA concentrations for 24 h did not cause XBP1 cleavage. b DU-145 cells were treated with 10 μM BA for varying time points or 1 μM thapsigargin (Tg) as a positive control. XBP1 was not cleaved at any time point by BA, but it was by Tg ( p

    Article Snippet: Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Positive Control

    BA stimulates ATF6 activation and translocation to the nucleus. a ATF6 ( green ) was present in the nucleus ( blue ) of DU-145 cells 1 ( p = 0.026, n = 17) and 2 h ( p = 0.002, n = 48) post-treatment with 10 μM BA. b ATF6 is activated by cleavage. The proportion of the cleaved product (p70) to full length (p100) was significantly elevated 30 min post-treatment ( p = 0.032, n = 3). One micromole of thapsigargin (Tg) also significantly increased the p70/p100 ratio ( p

    Journal: Biological Trace Element Research

    Article Title: Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

    doi: 10.1007/s12011-016-0824-y

    Figure Lengend Snippet: BA stimulates ATF6 activation and translocation to the nucleus. a ATF6 ( green ) was present in the nucleus ( blue ) of DU-145 cells 1 ( p = 0.026, n = 17) and 2 h ( p = 0.002, n = 48) post-treatment with 10 μM BA. b ATF6 is activated by cleavage. The proportion of the cleaved product (p70) to full length (p100) was significantly elevated 30 min post-treatment ( p = 0.032, n = 3). One micromole of thapsigargin (Tg) also significantly increased the p70/p100 ratio ( p

    Article Snippet: Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Translocation Assay

    BA induces an increase in GRP78 (BiP) protein in DU-145 cells. a DU-145 cells were treated with 10 μM BA for 0, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h. GAPDH was used as an internal loading control. GRP78/BiP translation was increased 0.5 ( p = 0.028, n = 4), 1 ( p = 0.007, n = 4), 2 h ( p = 0.032, n = 4), and 3 h ( p = 0.013, n = 4). b DU-145 cells treated with 1 μM thapsigargin (Tg) or DMSO (DM) vehicle for 1 h ( p

    Journal: Biological Trace Element Research

    Article Title: Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

    doi: 10.1007/s12011-016-0824-y

    Figure Lengend Snippet: BA induces an increase in GRP78 (BiP) protein in DU-145 cells. a DU-145 cells were treated with 10 μM BA for 0, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h. GAPDH was used as an internal loading control. GRP78/BiP translation was increased 0.5 ( p = 0.028, n = 4), 1 ( p = 0.007, n = 4), 2 h ( p = 0.032, n = 4), and 3 h ( p = 0.013, n = 4). b DU-145 cells treated with 1 μM thapsigargin (Tg) or DMSO (DM) vehicle for 1 h ( p

    Article Snippet: Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques:

    BA induces an increase in GADD34 protein in DU-145 cells. a DU-145 cells were treated with 10 μM BA for 0, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h. GADD34 was increased in cells at 2 ( p = 0.018, n = 3) and 3 h ( p = 0.011, n = 3). b DU-145 cells treated with 1 μM thapsigargin (Tg) or DMSO (DM) for 1 h, ( p

    Journal: Biological Trace Element Research

    Article Title: Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

    doi: 10.1007/s12011-016-0824-y

    Figure Lengend Snippet: BA induces an increase in GADD34 protein in DU-145 cells. a DU-145 cells were treated with 10 μM BA for 0, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h. GADD34 was increased in cells at 2 ( p = 0.018, n = 3) and 3 h ( p = 0.011, n = 3). b DU-145 cells treated with 1 μM thapsigargin (Tg) or DMSO (DM) for 1 h, ( p

    Article Snippet: Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques:

    Thapsigargin-dependent Ca 2+ entry in S2 cells. Fluo-4 fluorescence changes were monitored using a FLIPR 384 . After 20 s of recording, the 384-well pipette-tip head was lowered into the solution creating an offset artifact in the recording. This offset artifact is unrelated to a cellular response and is dependent on the fluid volume in each well at the start of the experiment and the extent of tip penetration into the solution. 10 s after lowering the pipette-tip head, either thapsigargin (TG, 1 μM final, circles) or DMSO (triangles) was injected (arrow). CaCl 2 was then added to achieve a final concentration of 1.8 mM. Traces were zeroed at time 0, and each data point represents the mean (±SEM) of 192 replicates.

    Journal: The Journal of General Physiology

    Article Title: A Store-operated Calcium Channel in Drosophila S2 Cells

    doi: 10.1085/jgp.200308982

    Figure Lengend Snippet: Thapsigargin-dependent Ca 2+ entry in S2 cells. Fluo-4 fluorescence changes were monitored using a FLIPR 384 . After 20 s of recording, the 384-well pipette-tip head was lowered into the solution creating an offset artifact in the recording. This offset artifact is unrelated to a cellular response and is dependent on the fluid volume in each well at the start of the experiment and the extent of tip penetration into the solution. 10 s after lowering the pipette-tip head, either thapsigargin (TG, 1 μM final, circles) or DMSO (triangles) was injected (arrow). CaCl 2 was then added to achieve a final concentration of 1.8 mM. Traces were zeroed at time 0, and each data point represents the mean (±SEM) of 192 replicates.

    Article Snippet: Initial fluorescence levels were recorded for 30 s, followed by addition of vehicle (0.01% DMSO) or 1 μM thapsigargin (LC Labs).

    Techniques: Fluorescence, Transferring, Injection, Concentration Assay

    Activation of Ca 2+ current by store depletion. Pipette solution 11 (Ca 2+ buffered to 310 nM). (A) Control currents with no activation of Ca 2+ current (note current scale). (B) IP 3 -activated Ca 2+ currents (10 μM IP 3 added to pipette), during exposure to 2 (□) or 20 (▪) mM Ca 2+ . Note complex changes in current during exposure to varying external Ca 2+ . (C) Thapsigargin (TG, 1 μM) applied externally at indicated time (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca 2+ . (D) Ionomycin (10 μM) applied externally at indicated times (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca 2+ .

    Journal: The Journal of General Physiology

    Article Title: A Store-operated Calcium Channel in Drosophila S2 Cells

    doi: 10.1085/jgp.200308982

    Figure Lengend Snippet: Activation of Ca 2+ current by store depletion. Pipette solution 11 (Ca 2+ buffered to 310 nM). (A) Control currents with no activation of Ca 2+ current (note current scale). (B) IP 3 -activated Ca 2+ currents (10 μM IP 3 added to pipette), during exposure to 2 (□) or 20 (▪) mM Ca 2+ . Note complex changes in current during exposure to varying external Ca 2+ . (C) Thapsigargin (TG, 1 μM) applied externally at indicated time (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca 2+ . (D) Ionomycin (10 μM) applied externally at indicated times (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca 2+ .

    Article Snippet: Initial fluorescence levels were recorded for 30 s, followed by addition of vehicle (0.01% DMSO) or 1 μM thapsigargin (LC Labs).

    Techniques: Activation Assay, Transferring

    Thapsigargin could not mimic the effects of tunicamycin on MDR GC cells. a Concentration-survival curves of GC cells treated with Tg or Tu (wide dose range) for 48 h. Tg, thapsigargin. b Expressions of UPR-related proteins in GC cells after Tg treatment (2 μg/ml) for 48 h determined by WB. All proteins were normalized to β-actin. c The effects of Tg on the chemosensitivity of GC cells after treatment for 48 h. ns, non-significant; * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Tunicamycin specifically aggravates ER stress and overcomes chemoresistance in multidrug-resistant gastric cancer cells by inhibiting N-glycosylation

    doi: 10.1186/s13046-018-0935-8

    Figure Lengend Snippet: Thapsigargin could not mimic the effects of tunicamycin on MDR GC cells. a Concentration-survival curves of GC cells treated with Tg or Tu (wide dose range) for 48 h. Tg, thapsigargin. b Expressions of UPR-related proteins in GC cells after Tg treatment (2 μg/ml) for 48 h determined by WB. All proteins were normalized to β-actin. c The effects of Tg on the chemosensitivity of GC cells after treatment for 48 h. ns, non-significant; * P

    Article Snippet: Thapsigargin (Tg, #12758) was from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Concentration Assay, Western Blot

    Synergism between ER stress and other stimuli. a FLS ( n = 4) were left untreated or stimulated with 1 μg/ml LPS or 1 ng/ml IL-1β in the presence or absence of 10 nM TG. Expression of IL6 and IL8 was measured by qPCR 4 h or 8 h after stimulation. b , c FLS ( n = 3, n = 2 for TG alone) were stimulated with 10 nM TG or 1 μg/ml LPS in the presence or absence of 10 nM TG for 8 h. Expression of 84 inflammatory genes was monitored by qPCR-based array. Heat map ( b ) depicts per-gene z-scores of log-scaled expression values relative to GAPDH, and bar graph ( c ) presents fold changes relative to unstimulated cells for 30 genes with the highest overall level of regulation (mean across all conditions). ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, IL Interleukin, LPS Lipopolysaccharide, qPCR Quantitative polymerase chain reaction, TG Thapsigargin

    Journal: Arthritis Research & Therapy

    Article Title: Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1186/s13075-017-1386-x

    Figure Lengend Snippet: Synergism between ER stress and other stimuli. a FLS ( n = 4) were left untreated or stimulated with 1 μg/ml LPS or 1 ng/ml IL-1β in the presence or absence of 10 nM TG. Expression of IL6 and IL8 was measured by qPCR 4 h or 8 h after stimulation. b , c FLS ( n = 3, n = 2 for TG alone) were stimulated with 10 nM TG or 1 μg/ml LPS in the presence or absence of 10 nM TG for 8 h. Expression of 84 inflammatory genes was monitored by qPCR-based array. Heat map ( b ) depicts per-gene z-scores of log-scaled expression values relative to GAPDH, and bar graph ( c ) presents fold changes relative to unstimulated cells for 30 genes with the highest overall level of regulation (mean across all conditions). ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, IL Interleukin, LPS Lipopolysaccharide, qPCR Quantitative polymerase chain reaction, TG Thapsigargin

    Article Snippet: ER stress was induced by tunicamycin from Streptomyces sp. (10 μg/ml; Sigma-Aldrich) or thapsigargin at varying concentrations (Calbiochem/Merck, Amsterdam-Zuidoost, The Netherlands).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effects of ER stress on gene transcription and mRNA stability in RA FLS. a FLS ( n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for the indicated amount of time. mRNA expression of mature and primary forms of transcripts of IL6 and IL8 was measured by qPCR. Shown is the ratio between expression observed in both experimental conditions at each time point. b FLS ( n = 8) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for 4 h, followed by incubation with 10 μg/ml ActD to induce transcriptional block. Cells were lysed at the indicated time points after addition of ActD, and the amount of transcript for each gene remaining in cells was analyzed by qPCR. Data are presented as a fraction of transcript detectable at each time point relative to the moment immediately after addition of ActD. Table shows 95% CIs of transcript half-life calculated using Prism software (GraphPad Software). ActD Actinomycin D; CXCL Chemokine (C-X-C motif) ligand, ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, IL Interleukin, LPS Lipopolysaccharide, mRNA Messenger RNA, qPCR Quantitative polymerase chain reaction, RA Rheumatoid arthritis, TG Thapsigargin

    Journal: Arthritis Research & Therapy

    Article Title: Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1186/s13075-017-1386-x

    Figure Lengend Snippet: Effects of ER stress on gene transcription and mRNA stability in RA FLS. a FLS ( n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for the indicated amount of time. mRNA expression of mature and primary forms of transcripts of IL6 and IL8 was measured by qPCR. Shown is the ratio between expression observed in both experimental conditions at each time point. b FLS ( n = 8) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for 4 h, followed by incubation with 10 μg/ml ActD to induce transcriptional block. Cells were lysed at the indicated time points after addition of ActD, and the amount of transcript for each gene remaining in cells was analyzed by qPCR. Data are presented as a fraction of transcript detectable at each time point relative to the moment immediately after addition of ActD. Table shows 95% CIs of transcript half-life calculated using Prism software (GraphPad Software). ActD Actinomycin D; CXCL Chemokine (C-X-C motif) ligand, ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, IL Interleukin, LPS Lipopolysaccharide, mRNA Messenger RNA, qPCR Quantitative polymerase chain reaction, RA Rheumatoid arthritis, TG Thapsigargin

    Article Snippet: ER stress was induced by tunicamycin from Streptomyces sp. (10 μg/ml; Sigma-Aldrich) or thapsigargin at varying concentrations (Calbiochem/Merck, Amsterdam-Zuidoost, The Netherlands).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Blocking Assay, Software

    Synergism between ER stress and LPS in dermal fibroblasts and macrophages. a , c Human dermal fibroblasts ( a , n = 3) or GM-CSF-differentiated macrophages ( c , n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for the indicated amount of time. mRNA expression of mature and primary forms of transcripts of IL6 , IL8 , and TNF was measured by qPCR. Shown is the ratio between expression observed in both experimental conditions at each time point. b , d Human dermal fibroblasts ( b , n = 3) or GM-CSF-differentiated macrophages ( d , n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for 4 h, followed by incubation with 10 μg/ml ActD to induce transcriptional block. Cells were lysed at the indicated time points after addition of ActD, and the amount of transcript for each gene remaining in cells was analyzed by qPCR. Data are presented as a fraction of transcript detectable at each time point relative to the moment immediately after addition of ActD. ActD Actinomycin D, ER Endoplasmic reticulum, GM-CSF Granulocyte-macrophage colony-stimulating factor, IL Interleukin, LPS Lipopolysaccharide, mRNA Messenger RNA, qPCR Quantitative polymerase chain reaction, TG Thapsigargin, TNF Tumor necrosis factor

    Journal: Arthritis Research & Therapy

    Article Title: Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.1186/s13075-017-1386-x

    Figure Lengend Snippet: Synergism between ER stress and LPS in dermal fibroblasts and macrophages. a , c Human dermal fibroblasts ( a , n = 3) or GM-CSF-differentiated macrophages ( c , n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for the indicated amount of time. mRNA expression of mature and primary forms of transcripts of IL6 , IL8 , and TNF was measured by qPCR. Shown is the ratio between expression observed in both experimental conditions at each time point. b , d Human dermal fibroblasts ( b , n = 3) or GM-CSF-differentiated macrophages ( d , n = 3) were stimulated with 1 μg/ml LPS in the presence or absence of 10 nM TG for 4 h, followed by incubation with 10 μg/ml ActD to induce transcriptional block. Cells were lysed at the indicated time points after addition of ActD, and the amount of transcript for each gene remaining in cells was analyzed by qPCR. Data are presented as a fraction of transcript detectable at each time point relative to the moment immediately after addition of ActD. ActD Actinomycin D, ER Endoplasmic reticulum, GM-CSF Granulocyte-macrophage colony-stimulating factor, IL Interleukin, LPS Lipopolysaccharide, mRNA Messenger RNA, qPCR Quantitative polymerase chain reaction, TG Thapsigargin, TNF Tumor necrosis factor

    Article Snippet: ER stress was induced by tunicamycin from Streptomyces sp. (10 μg/ml; Sigma-Aldrich) or thapsigargin at varying concentrations (Calbiochem/Merck, Amsterdam-Zuidoost, The Netherlands).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Blocking Assay

    SDF-1 and the GLP-1 receptor agonist EXD4 act additively to preserve and enhance beta cell mass. INS-1 cells were cultured in 96 well plates in the presence of 2 nmol/l EXD4 and/or 10 nmol/l SDF-1 on a background of ( a ) serum deprivation or ( b ) serum repletion (0.8%). EXD4 and SDF-1 were replenished every 2 days. Cell viability was measured 6 days after treatment by ATPlite assay. INS-1 cells treated with ( c ) thapsigargin or ( d ) cytokines were incubated with vehicle or 10 nmol/l SDF-1, 10 nmol/l EXD4 or SDF-1+EXD4 for 6 days and their dry weight was then measured. Data are expressed as mass relative to the mass for vehicle treatment. e INS-1 cells were cultured in 96 well plates in the presence of 10 nmol/l SDF-1 with or without AMD3100 (25 μmol/l) on a background of nutrient deprivation by serum withdrawal (no serum). f INS-1 cells were cultured in 96 well plates in the presence of increasing doses of SDF-1 on a background of normal (5 mmol/l) or high glucose concentration (25 mmol/l). Reagents were added at day 0 and day 2, and 4 days after treatment, cell viability was measured using the ATPlite assay. g Dispersed human islets were cultured in 96 well plates (~100 islets/well) in the presence of 2 nmol/l EXD4, and/or 10 nmol/l SDF-1 on a background of cytokines (IL-1β 50 ng/ml, TNFα 10 ng/ml, IFNγ 50 ng/ml). Cell viability was measured 3 days after treatment by ATPlite assay. The viable beta cell number was enhanced additively by EXD4 and SDF-1. Therefore the combination of GLP-1 and SDF-1 increased human islet cell viability against cytokine-stress-induced cell death. h For the insulin secretion assay, INS-1 cells were plated in 96 well plates. SDF-1 (2 nmol/l) and AMD3100 were added at day 0, and the insulin concentration of culture medium was measured at day 6. Statistical significance is depicted as * p

    Journal: Diabetologia

    Article Title: Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival

    doi: 10.1007/s00125-011-2181-x

    Figure Lengend Snippet: SDF-1 and the GLP-1 receptor agonist EXD4 act additively to preserve and enhance beta cell mass. INS-1 cells were cultured in 96 well plates in the presence of 2 nmol/l EXD4 and/or 10 nmol/l SDF-1 on a background of ( a ) serum deprivation or ( b ) serum repletion (0.8%). EXD4 and SDF-1 were replenished every 2 days. Cell viability was measured 6 days after treatment by ATPlite assay. INS-1 cells treated with ( c ) thapsigargin or ( d ) cytokines were incubated with vehicle or 10 nmol/l SDF-1, 10 nmol/l EXD4 or SDF-1+EXD4 for 6 days and their dry weight was then measured. Data are expressed as mass relative to the mass for vehicle treatment. e INS-1 cells were cultured in 96 well plates in the presence of 10 nmol/l SDF-1 with or without AMD3100 (25 μmol/l) on a background of nutrient deprivation by serum withdrawal (no serum). f INS-1 cells were cultured in 96 well plates in the presence of increasing doses of SDF-1 on a background of normal (5 mmol/l) or high glucose concentration (25 mmol/l). Reagents were added at day 0 and day 2, and 4 days after treatment, cell viability was measured using the ATPlite assay. g Dispersed human islets were cultured in 96 well plates (~100 islets/well) in the presence of 2 nmol/l EXD4, and/or 10 nmol/l SDF-1 on a background of cytokines (IL-1β 50 ng/ml, TNFα 10 ng/ml, IFNγ 50 ng/ml). Cell viability was measured 3 days after treatment by ATPlite assay. The viable beta cell number was enhanced additively by EXD4 and SDF-1. Therefore the combination of GLP-1 and SDF-1 increased human islet cell viability against cytokine-stress-induced cell death. h For the insulin secretion assay, INS-1 cells were plated in 96 well plates. SDF-1 (2 nmol/l) and AMD3100 were added at day 0, and the insulin concentration of culture medium was measured at day 6. Statistical significance is depicted as * p

    Article Snippet: Thapsigargin was obtained from Biomol (Ply-mouth Meeting, PA, USA).

    Techniques: Activated Clotting Time Assay, Cell Culture, Incubation, Concentration Assay

    Beta cell injury induces expression of Sdf1 in islets. Sdf1 mRNA levels are induced in ( a ) mouse and ( b ) human islets by beta cell stress-activating agents. a Cytokines (50 ng/ml IL-1β, 10 ng/ml TNFα, 50 ng/ml IFNγ) and 50 nmol/l thapsigargin were added to mouse islets. b ]. All values are expressed relative to the value of the control-treated cells. Thaps, thapsigargin

    Journal: Diabetologia

    Article Title: Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival

    doi: 10.1007/s00125-011-2181-x

    Figure Lengend Snippet: Beta cell injury induces expression of Sdf1 in islets. Sdf1 mRNA levels are induced in ( a ) mouse and ( b ) human islets by beta cell stress-activating agents. a Cytokines (50 ng/ml IL-1β, 10 ng/ml TNFα, 50 ng/ml IFNγ) and 50 nmol/l thapsigargin were added to mouse islets. b ]. All values are expressed relative to the value of the control-treated cells. Thaps, thapsigargin

    Article Snippet: Thapsigargin was obtained from Biomol (Ply-mouth Meeting, PA, USA).

    Techniques: Expressing

    Flx affects osteoclastogenesis in a 5HTT-independent manner ( a ) Quantification of TRAP activity (left) and gene expression in primary osteoclast cultures (OCs) derived from 5HTT-deficient ( Slc6a4 −/− ) mice. Ctsk, Cathepsin K. Clcn7, Chloride channel 7. Acp5 encodes TRAP. ( b ) Representative images ( n = 4 images/mouse) of vertebrae (left) from Slc6a4 −/− females treated for 3 w (veh n = 8, Flx n = 6). Quantification of BV/TV is indicated below each image. Scale bars, 200 μm. Bone histomorphometry of these mice is also indicated (middle and right). ( c , d ) Gene expression in WT OCs ( c ) and Slc6a4 −/− OCs ( d ). Mitf encodes microphthalmia-associated transcription factor; Undiff., undifferentiated OCs. ( e ) Expression of Nfatc1 in long bones of WT females treated for 3 w. ( f ) Western blot (left) and quantification of band intensities reported to veh (right) of indicated proteins in RAW264.7 osteoclasts treated with Flx or veh ( n = 1 technical replicate of the number of biological replicates indicated in the bar graph (left)). ( g ) Representative curves ( n = 13) of Fura-2 ratio (340/380) in individual RAW264.7 osteoclasts recorded in veh medium and after addition of Flx (left), Fura-2 ratio levels after Flx addition relative to veh (AUC, area under the curve ( n = 13) (middle), and proportion of OCs and MNCs responding to Flx (right). Thap, Thapsigargin. ( h ) Gene expression in RAW264.7 osteoclasts treated for 24 h. ( i ) Gene expression in Creb fl/fl:virus-CRE OCs treated for 6 days (6 d) (left), and the percentage of resorbed area in a pit resorption assay of WT OCs treated for 24 h with W5, KG-501 and Flx (right). ( j ) Gene expression in spleen of WT or Slc6a4 −/− females treated for 3 weeks. Rorc encodes RORγt. Values are mean ± SEM compared to veh (*) or undiff. OCs ( $ ) */ $ P ≤ 0.05, ** P ≤ 0.01, ***/ $$$ P ≤ 0.001, ****/ $$$$ P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test ( a , i ), one-way ANOVA followed by Turkey’s ( c , d , h ) or Student’s test (all other panels).

    Journal: Nature medicine

    Article Title: Serotonin reuptake inhibitors act centrally to cause bone loss in mice by counteracting a local antiresorptive effect

    doi: 10.1038/nm.4166

    Figure Lengend Snippet: Flx affects osteoclastogenesis in a 5HTT-independent manner ( a ) Quantification of TRAP activity (left) and gene expression in primary osteoclast cultures (OCs) derived from 5HTT-deficient ( Slc6a4 −/− ) mice. Ctsk, Cathepsin K. Clcn7, Chloride channel 7. Acp5 encodes TRAP. ( b ) Representative images ( n = 4 images/mouse) of vertebrae (left) from Slc6a4 −/− females treated for 3 w (veh n = 8, Flx n = 6). Quantification of BV/TV is indicated below each image. Scale bars, 200 μm. Bone histomorphometry of these mice is also indicated (middle and right). ( c , d ) Gene expression in WT OCs ( c ) and Slc6a4 −/− OCs ( d ). Mitf encodes microphthalmia-associated transcription factor; Undiff., undifferentiated OCs. ( e ) Expression of Nfatc1 in long bones of WT females treated for 3 w. ( f ) Western blot (left) and quantification of band intensities reported to veh (right) of indicated proteins in RAW264.7 osteoclasts treated with Flx or veh ( n = 1 technical replicate of the number of biological replicates indicated in the bar graph (left)). ( g ) Representative curves ( n = 13) of Fura-2 ratio (340/380) in individual RAW264.7 osteoclasts recorded in veh medium and after addition of Flx (left), Fura-2 ratio levels after Flx addition relative to veh (AUC, area under the curve ( n = 13) (middle), and proportion of OCs and MNCs responding to Flx (right). Thap, Thapsigargin. ( h ) Gene expression in RAW264.7 osteoclasts treated for 24 h. ( i ) Gene expression in Creb fl/fl:virus-CRE OCs treated for 6 days (6 d) (left), and the percentage of resorbed area in a pit resorption assay of WT OCs treated for 24 h with W5, KG-501 and Flx (right). ( j ) Gene expression in spleen of WT or Slc6a4 −/− females treated for 3 weeks. Rorc encodes RORγt. Values are mean ± SEM compared to veh (*) or undiff. OCs ( $ ) */ $ P ≤ 0.05, ** P ≤ 0.01, ***/ $$$ P ≤ 0.001, ****/ $$$$ P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test ( a , i ), one-way ANOVA followed by Turkey’s ( c , d , h ) or Student’s test (all other panels).

    Article Snippet: We drawn regions of interest (ROI) around osteoclasts and mononuclear cells, and images were acquired using a 40X 1.3 NA oil objective for a total of 8 min. We added 3 μM of fluoxetine (Flx) with a pipette at t = 3 min and 3 μM sarco/endoplasmics reticulum ATPase blocker, Thapsigargin (Thap) (Sigma, St. Louis, Missouri) at t = 6 min. We used increased cytosolic Ca2+ concentration by Thap as a positive control of cell viability.

    Techniques: Activity Assay, Expressing, Derivative Assay, Mouse Assay, Western Blot

    A CK2 inhibitor inhibited ER stress-induced GRP78 expression. 4,5,6,7-tetrabromobenzotriazole (TBB) inhibited GRP78 expression at the mRNA and protein levels. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. RT-PCR was performed using specific primers for each mRNA. n = 4/group * p

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: A CK2 inhibitor inhibited ER stress-induced GRP78 expression. 4,5,6,7-tetrabromobenzotriazole (TBB) inhibited GRP78 expression at the mRNA and protein levels. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and then treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. RT-PCR was performed using specific primers for each mRNA. n = 4/group * p

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    The CK2 inhibitor did not affect the eIF2α-CHOP arm of the ER stress-induced UPR. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 4 h. Western blotting was then performed. TBB did not affect ER stress-induced eIF2α phosphorylation. Densitometric analysis of the normalization data of the phospho-eIF2α and eIF2α intensities were done. n = 3 per group. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. A RT-PCR analysis was then performed. n = 3/group TBB did not affect ER stress-induced CHOP mRNA expression. (C) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h. Western blotting was then performed. n = 4/group TBB did not affect ER stress-induced CHOP protein production. Results are expressed as the means ± S.E.

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: The CK2 inhibitor did not affect the eIF2α-CHOP arm of the ER stress-induced UPR. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 4 h. Western blotting was then performed. TBB did not affect ER stress-induced eIF2α phosphorylation. Densitometric analysis of the normalization data of the phospho-eIF2α and eIF2α intensities were done. n = 3 per group. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. A RT-PCR analysis was then performed. n = 3/group TBB did not affect ER stress-induced CHOP mRNA expression. (C) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h. Western blotting was then performed. n = 4/group TBB did not affect ER stress-induced CHOP protein production. Results are expressed as the means ± S.E.

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

    CK2 inhibitor did not affect ER stress-induced ATF6 processing. (A) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 18 h and Western blotting analysis was performed. TBB inhibited ER stress-induced GRP78 expression. * p

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: CK2 inhibitor did not affect ER stress-induced ATF6 processing. (A) SH-SY5Y cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, and then treated with thapsigargin (Tg: 10 µM) for 18 h and Western blotting analysis was performed. TBB inhibited ER stress-induced GRP78 expression. * p

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Western Blot, Expressing

    ER stress did not affect expression of CK2. (A) Primary cultured glial cells were treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h and subjected to Western blotting. ER stress increased GRP78 levels whereas it did not affect levels of CK2. (B) Densitometric analysis of expression of CK2. n = 4/group. Results are expressed as the means ± S.E.

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: ER stress did not affect expression of CK2. (A) Primary cultured glial cells were treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h and subjected to Western blotting. ER stress increased GRP78 levels whereas it did not affect levels of CK2. (B) Densitometric analysis of expression of CK2. n = 4/group. Results are expressed as the means ± S.E.

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Expressing, Cell Culture, Western Blot

    The CK2 inhibitor inhibited ER stress-induced XBP-1 mRNA splicing. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h, and subjected to a RT-PCR analysis. (B) Densitometric analysis of spliced/unspliced XBP-1 mRNA. An ER stress inducer increased XBP-1 splicing and this effect was significantly inhibited by TBB. n = 4–5/group * p

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: The CK2 inhibitor inhibited ER stress-induced XBP-1 mRNA splicing. (A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h, and subjected to a RT-PCR analysis. (B) Densitometric analysis of spliced/unspliced XBP-1 mRNA. An ER stress inducer increased XBP-1 splicing and this effect was significantly inhibited by TBB. n = 4–5/group * p

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Knocking down CK2 inhibited ER stress-induced GRP78 expression. (A) Western blot analysis of endogenous CK2α expression in lysates of nontransfected cells (NT) and cells transfected with (short interfering RNA) siRNAs (75 nM) directed at CK2 or the control sequence. CK2 siRNA reduced the expression of CK2 compared with NT or control siRNA. (B) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA and treated with tunicamycin (Tm: 0.01 µg/mL) for 4 h. RT-PCR was then performed. n = 3/group (C, D) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h, and subjected to Western blotting. n = 3–4/group * p

    Journal: PLoS ONE

    Article Title: Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

    doi: 10.1371/journal.pone.0040144

    Figure Lengend Snippet: Knocking down CK2 inhibited ER stress-induced GRP78 expression. (A) Western blot analysis of endogenous CK2α expression in lysates of nontransfected cells (NT) and cells transfected with (short interfering RNA) siRNAs (75 nM) directed at CK2 or the control sequence. CK2 siRNA reduced the expression of CK2 compared with NT or control siRNA. (B) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA and treated with tunicamycin (Tm: 0.01 µg/mL) for 4 h. RT-PCR was then performed. n = 3/group (C, D) CK2 siRNA decreased ER stress-induced GRP78 expression compared with control siRNA. Primary cultured glial cells were transfected with 75 nM siRNA, treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h, and subjected to Western blotting. n = 3–4/group * p

    Article Snippet: Materials Tunicamycin (Tm) and thapsigargin (Tg) were obtained from Wako Pure Chemical Ltd. (Japan).

    Techniques: Expressing, Western Blot, Transfection, Small Interfering RNA, Sequencing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    NAADP-elicited Ca 2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice ( n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice ( n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin ( n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca 2+ responses to NAADP (100 nM), IP 3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 ( n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.

    Journal: Cell Calcium

    Article Title: Both RyRs and TPCs are required for NAADP-induced intracellular Ca2+ release

    doi: 10.1016/j.ceca.2015.05.005

    Figure Lengend Snippet: NAADP-elicited Ca 2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice ( n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice ( n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin ( n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca 2+ responses to NAADP (100 nM), IP 3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 ( n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.

    Article Snippet: Thapsigargin and ATP were from Merck Millipore (UK).

    Techniques: Isolation, Mouse Assay, Incubation

    Involvement of TPC2 channels in NAADP-elicited Ca 2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse ( n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse ( n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP ( n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) ( n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP ( n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP 3 ( n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR ( n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca 2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.

    Journal: Cell Calcium

    Article Title: Both RyRs and TPCs are required for NAADP-induced intracellular Ca2+ release

    doi: 10.1016/j.ceca.2015.05.005

    Figure Lengend Snippet: Involvement of TPC2 channels in NAADP-elicited Ca 2+ release. (A) Trace represents an application of 100 nM NAADP to permeabilized cell isolated from wt mouse ( n = 13). (B) Representative trace of application of 100 nM NAADP to permeabilized cell isolated from TPC2-KO mouse ( n = 5). (C) Permeabilized cells from wt mice were pre-incubated with TPC2 antibody (20 min, 1:100) followed by addition of 100 nM NAADP ( n = 8). (D) Representative trace shows typical response to 100 nM NAADP in permeabilized cells from TPC2-KO mice treated with TPC1 antibody (20 min, 1:100) ( n = 11). (E) Comparison of the amplitudes of responses to 100 nM NAADP ( n = 13 control; n = 8 with TPC2 antibody treatment), 10 μM IP 3 ( n = 9 control; n = 8 with TPC2 antibody treatment) or 10 μM cADPR ( n = 9 control; n = 5 with TPC2 antibody treatment) in pre-treated or non-treated cells with antibody against TPC2. (F) Summary of NAADP-elicited Ca 2+ release from the intracellular stores in permeabilized control cells or pre-treated with antibodies against TPC1 (20.6 ± 0.7%, n = 11), or TPC2 (7.6 ± 1%, SEM, n = 8), or mixture of both (5.3 ± 0.2%, n = 4), in wt mice compared to responses in permeabilized cells isolated from TPC2 KO mice and treated (3.9 ± 0.3%, n = 7) or non-treated with TPC1 antibody (9.8 ± 1.0%, n = 5). (G) Summary of results obtained from permeabilized cells treated with 10 μM thapsigargin to deplete ER followed by subsequent application of NAADP (100 nM) in the presence of antibodies against TPC1 (9.8 ± 0.5%, n = 5, p > 0.08), or TPC2 (1.63 ± 0.3%, n = 6), or a mixture of both (0.83 ± 0.1%, n = 6), or a mixture of antibodies against RyR1 and RyR3 (0.75 ± 0.1%, n = 5) as compared to control NAADP responses (12.4 ± 1.1%, n = 5). Error bars represent ± SEM. Cells were loaded with Fluo-5N in AM form.

    Article Snippet: Thapsigargin and ATP were from Merck Millipore (UK).

    Techniques: Isolation, Mouse Assay, Incubation

    Expression of bbc3/PUMA may be sufficient and required for ER stress–induced apoptosis in human cells. (A) Transient transfection of SH-SY5Y neuroblastoma cells with bbc3/PUMA leads to induction of apoptosis. Cells were cotransfected with expression plasmids encoding enhanced GFP (pEGFP) and either a hemagglutinin-tagged (HA) version of bbc3/PUMA (pHA-PUMA) or empty vector (pcDNA3.1). 12 h after transfection, cells were fixed and stained with Hoechst 33258. Arrowheads point to transfected cells with the apoptotic phenotype. Similar results were obtained in a separate experiment. (B) Quantitative FACS ® analysis of PI uptake of EGFP-positive SH-SY5Y cells transiently cotransfected with expression vectors encoding hemagglutinin-tagged (HA) versions of Bbc3/PUMA, Bbc3/PUMA-ΔBH3, or empty vector (pcDNA3.1). Data were normalized to EGFP-only transfected controls and represent 10,000 events each. For evaluation of comparable transfection rates, whole-cell extracts of cells harvested 16 h after transfection were analyzed by Western blotting. Blots were probed with an anti-hemagglutinin or anti- α-tubulin antibody as loading control. (C) Protein levels of BiP, Bbc3/PUMA, and α-tubulin in human HCT116 control and Bbc3/PUMA-deficient cells treated with 1 μM thapsigargin or vehicle for the indicated time points. (D) FACS ® analysis of sub-G1 cells of human HCT116 control and Bbc3/PUMA-deficient cells exposed to 1 μM thapsigargin (TH) or vehicle for 36 h. (E) Quantitative FACS ® analysis of thapsigargin- and STS (3 μM)-induced apoptosis. Data from 10,000 events each are means ± SEM from n = 3 separate experiments per time point.

    Journal: The Journal of Cell Biology

    Article Title: Gene expression during ER stress-induced apoptosis in neurons

    doi: 10.1083/jcb.200305149

    Figure Lengend Snippet: Expression of bbc3/PUMA may be sufficient and required for ER stress–induced apoptosis in human cells. (A) Transient transfection of SH-SY5Y neuroblastoma cells with bbc3/PUMA leads to induction of apoptosis. Cells were cotransfected with expression plasmids encoding enhanced GFP (pEGFP) and either a hemagglutinin-tagged (HA) version of bbc3/PUMA (pHA-PUMA) or empty vector (pcDNA3.1). 12 h after transfection, cells were fixed and stained with Hoechst 33258. Arrowheads point to transfected cells with the apoptotic phenotype. Similar results were obtained in a separate experiment. (B) Quantitative FACS ® analysis of PI uptake of EGFP-positive SH-SY5Y cells transiently cotransfected with expression vectors encoding hemagglutinin-tagged (HA) versions of Bbc3/PUMA, Bbc3/PUMA-ΔBH3, or empty vector (pcDNA3.1). Data were normalized to EGFP-only transfected controls and represent 10,000 events each. For evaluation of comparable transfection rates, whole-cell extracts of cells harvested 16 h after transfection were analyzed by Western blotting. Blots were probed with an anti-hemagglutinin or anti- α-tubulin antibody as loading control. (C) Protein levels of BiP, Bbc3/PUMA, and α-tubulin in human HCT116 control and Bbc3/PUMA-deficient cells treated with 1 μM thapsigargin or vehicle for the indicated time points. (D) FACS ® analysis of sub-G1 cells of human HCT116 control and Bbc3/PUMA-deficient cells exposed to 1 μM thapsigargin (TH) or vehicle for 36 h. (E) Quantitative FACS ® analysis of thapsigargin- and STS (3 μM)-induced apoptosis. Data from 10,000 events each are means ± SEM from n = 3 separate experiments per time point.

    Article Snippet: Materials Tunicamycin, thapsigargin, etoposide, and STS were purchased from Qbiogene.

    Techniques: Expressing, Transfection, Plasmid Preparation, Staining, FACS, Western Blot

    Induction of Bbc3/PUMA is a general cellular response to prolonged ER stress. (A) Induction of bb33/PUMA mRNA in human SH-SY5Y neuroblastoma cells in response to thapsigargin (TH; 1 μM). Controls (Con) were treated with vehicle (DMSO, 24 h). (B) Bbc3/PUMA protein is expressed in primary hippocampal neurons after tunicamycin treatment. Cultures were treated for 24 h with 3 μM tunicamycin or vehicle. An unspecific band of ∼24 kD served as loading control. (C) Top, increased expression of the ER stress–specific transcription factor CHOP detected by immunohistochemistry in the selective vulnerable CA1 hippocampal subfield of rats subjected to transient forebrain ischemia (Isch). Increased expression of CHOP was detectable as early as 1 h postischemia. Bottom, expression of Bbc3/PUMA is increased in dying CA1 hippocampal pyramidal neurons 5 d after transient forebrain ischemia. Sham-operated animals served as respective controls. Bar, 25 μm. (D) Western blot analysis of Bbc3/PUMA expression in hippocampus and neocortex tissue homogenates 72 h after transient forebrain ischemia (Isch) or sham surgery (Con). Similar results were obtained in a second experiment.

    Journal: The Journal of Cell Biology

    Article Title: Gene expression during ER stress-induced apoptosis in neurons

    doi: 10.1083/jcb.200305149

    Figure Lengend Snippet: Induction of Bbc3/PUMA is a general cellular response to prolonged ER stress. (A) Induction of bb33/PUMA mRNA in human SH-SY5Y neuroblastoma cells in response to thapsigargin (TH; 1 μM). Controls (Con) were treated with vehicle (DMSO, 24 h). (B) Bbc3/PUMA protein is expressed in primary hippocampal neurons after tunicamycin treatment. Cultures were treated for 24 h with 3 μM tunicamycin or vehicle. An unspecific band of ∼24 kD served as loading control. (C) Top, increased expression of the ER stress–specific transcription factor CHOP detected by immunohistochemistry in the selective vulnerable CA1 hippocampal subfield of rats subjected to transient forebrain ischemia (Isch). Increased expression of CHOP was detectable as early as 1 h postischemia. Bottom, expression of Bbc3/PUMA is increased in dying CA1 hippocampal pyramidal neurons 5 d after transient forebrain ischemia. Sham-operated animals served as respective controls. Bar, 25 μm. (D) Western blot analysis of Bbc3/PUMA expression in hippocampus and neocortex tissue homogenates 72 h after transient forebrain ischemia (Isch) or sham surgery (Con). Similar results were obtained in a second experiment.

    Article Snippet: Materials Tunicamycin, thapsigargin, etoposide, and STS were purchased from Qbiogene.

    Techniques: Expressing, Immunohistochemistry, Western Blot