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  • 99
    Thermo Fisher thapsigargin
    Extracellular rhHSP70 induces intracellular Ca 2+ mobilization A. rhHSP70 induced Ca 2+ release from internal stores in HMEC. Data are expressed as the 340/380 nm excitation ratio in one cell to observe oscillations in [Ca 2+ ]i because the oscillatory process is not synchronized in cells of the same monolayer. External additions of drugs are indicated by arrows. Changes in external calcium bath conditions are indicated on the bottom of traces. In most cases, drugs were initially applied in the absence (0 Ca) then in Ca 2+ (1.8 mM) containing solution to reveal Ca 2+ release from internal stores then external Ca 2+ entry, respectively (representative from 50 cells; n=10). No calcium increase was induced by the cell superfusion of the control bath solution (middle trace) while <t>thapsigargin</t> (TSG 4μM) always produced a drastic increase in [Ca 2+ ]i (lower trace; Representative from 50 cells; n=4). B. Contribution of EGFR to rhHSP70-induced Ca 2+ signaling. Superimposed traces from cells preincubated with the EGFR (ErbB1) inhibitor, gefitinib (10 μM for 30 min; in red), before the addition of rhHSP70 (Representative from 50 cells; n=5). C. Effects of phospholipase C (PLC) inhibitor U-73122 (5 μM) and its inactive analog U-73343 (5 μM) on the rhHSP70-induced Ca 2+ oscillations. Drugs were applied without (0 Ca) then with extracellular Ca 2+ (1.8 mM) (representative from 50 cells; n=5). D. Ca 2+ oscillations required both Ca 2+ release from internal stores and store operated Ca 2+ entry (SOCE). Cells were pretreated with the selective SOCE inhibitor, BTP-2 (20 μM; 20 min), before challenged with rhHSP70 (Representative from 30 cells; n=4).
    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
    Activation of ER stress by human oxidized LDL. (a) Western blotting analysis comparing changes in PERK, eIF2 and Ire1α and their phosphorylated forms (p). Total proteins were prepared from MIN6 cells cultured with 2 mmol/l cholesterol oxidized LDL (oxLDL) for the indicated times and 1 μmol/l <t>thapsigargin</t> (Thaps) for 6 h. The α-tubulin protein served as loading control. The figure is a representative experiment out of three. Measurement of CHOP / Chop , P58IPK / p58IPK and ATF4 / Atf4 mRNA levels in ( b) and (d) MIN6 and ( c) and (e) isolated human islets cells cultured with oxidized LDL. The mRNA level was quantified by quantitative real-time PCR in MIN6 or isolated human islets cells cultured for 48 h with vehicle (V), native (nLDL) or oxidized LDL (oxLDL). The PBA chemical chaperone 2.5 mmol/l were added in the cells cultured with oxidized LDL (oxLDL, filled bar ). For d ) and e ), cells were cultured with oxidized LDL plus 1 mmol/l cholesterol HDL ( filled bar ). The mRNA level was normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene ( RPLP0 / Rplp0 ) and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P
    Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris thapsigargin
    GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with <t>thapsigargin</t> abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.
    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
    C/EBPβ modulates EBV lytic gene expression and replication by ER stressors . EBV + Akata cells and Rael were treated with 20nM bortezomib (BZ), 1μM <t>thapsigargin</t> (TG), or 2 μg/mL tunicamycin (TU) for 24 hours. (A) RNA was isolated
    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
    Disruption of lysosomal Ca 2+ uptake with bafilomycin A 1 does not affect SOCE. ( A ) Transient increase in [Ca 2+ ] i evoked by <t>thapsigargin</t> (1 µM, solid bar) in HEK cells in nominally Ca 2+ -free HBS in control and bafilomycin A 1 -treated cells (Baf A 1 ; 1 µM, 1 h). ( B ) Summary results show effects of bafilomycin A 1 (1 µM, 1 h) on the peak increase in [Ca 2+ ] i evoked by thapsigargin (1 µM) in nominally Ca 2+ -free HBS. Results are means ± s.e.m. from three independent experiments. ( C ) Restoration of extracellular Ca 2+ (30 mM) to cells pre-treated with thapsigargin (1 µM, 15 min) in nominally Ca 2+ -free HBS to deplete intracellular Ca 2+ stores evokes SOCE. The response is indistinguishable in control cells and cells treated with bafilomycin A 1 (1 µM, 1 h). ( D ) SOCE after restoration of different concentrations of extracellular Ca 2+ ([Ca 2+ ] e ) to control and bafilomycin A 1 -treated cells (1 µM, 1 h). Results are means ± s.e.m. from three independent experiments. ( E ) TPEN (100 µM, 2 min) in nominally Ca 2+ -free HBS was used to reduce the free [Ca 2+ ] within the ER before restoration of the indicated concentrations of extracellular Ca 2+ to control or bafilomycin A 1 -treated cells (1 µM, 1 h). Results show the peak increase in [Ca 2+ ] i detected within 60 s after restoration of extracellular Ca 2+ . ( F ) Peak increase in [Ca 2+ ] i evoked by CCh (1 mM) in the presence of TPEN (100 µM, 2 min) with and without pre-incubation with bafilomycin A 1 . * P
    Thapsigargin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical thapsigargin
    Orlistat-induced ER stress causes synergism with gemcitabine (A) (B) Western blot analyses of ER stress-related protein levels in PANC-1 cells with indicated treatments at 48 hours post-treatment. (C) Isobologram showing the combination index of gemcitabine and <t>thapsigargin</t> in three different schedules: simultaneous, sequential, and reverse sequential in PANC-1. Combination index was calculated from MTT data using Compusyn software. (D) Relative percentage of PANC-1 cells dual positive for CD24/CD44 stem cell markers upon treatment with control, and thapsigargin for 48 hours. Gem: Gemcitabine, Thap: Thapsigargin. ** P ≤ 0.01, compared to the control by two sample Student’s t-test. (E) Kaplan-Meier survival comparisons between top and bottom tertiles for ER stress marker gene enrichment in TCGA patient tumor specimens. Comparisons are made for all stages (n = 20) and stage II only (n = 18) pancreatic ductal adenocarcinoma patients treated with gemcitabine only by utilizing Gehan-Breslow-Wilcoxon Test.
    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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    96
    Abcam thapsigargin
    PERK is required for B7H6 upregulation under ER stress conditions. B7H6 surface levels were assessed by flow cytometry after treatment with 0.125 μg/ml <t>thapsigargin</t> (Tg) or mock treated with DMSO for 16 h in the following conditions: a 624 wt, PERK knockout (KO), IRE1 KO and PERK/IRE1 double KO (DKO) cells, to the right appears quantification of the average mean fluorescence intensity (MFI) ± STD of treated relative to untreated cells of three independent experiments. b Melanoma 526 wt and PERK KO cells. c 624 wt cells pretreated with 1 μM GSK or 0.5 μM ISRIB for 1 h. The lower panel shows quantification of the average MFI ± STD of treated relative to untreated cells of three independent experiments. d 624 CHOP KO cells. BG indicates secondary only background staining, which was similar for both treated and untreated cells (shown is the BG for untreated cells)
    Thapsigargin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM thapsigargin
    The effect of DLPhtEtn on PC-12 cell death induced by amyloid-β 1-40 peptide or <t>thapsigargin</t> . ( A ) PC-12 cells were treated with amyloid-β 1-40 peptide (5 μM) in the absence (Control) and presence of phospholipids (30 μM) as indicated in serum-free extracellular solution for 48 h, and cell viability was assayed. Data represents the mean (± SEM) percentage of basal levels (MTT intensities of cells untreated with amyloid-β 1-40 peptide) (n = 6 independent experiments). ** P
    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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    93
    Santa Cruz Biotechnology thapsigargin
    Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM <t>Thapsigargin</t> or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.
    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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    95
    Millipore 2 4 6 trihydroxyacetophenone thap
    Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM <t>Thapsigargin</t> or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.
    2 4 6 Trihydroxyacetophenone Thap, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories thapsigargin
    Pharmacology of IP 3 R response of local uncaging of caged Ca 2+ in CF15 cells in absence of extracellular Ca 2+ . A Example of line-scan images acquired at 2 ms per line and 0.21 μm per pixel in CF15 cells untreated at 37°C in presence or not of 100 μM 2-APB, 100 μM decavanadate, 20 mM caffeine or 10 μM cyclosporine A (all were preincubated during 10 min) and after 2 h incubation with 10 μM <t>thapsigargin</t> (TG). B Average of the line-scan images in A expressed as normalized fluorescence in each conditions C Mean normalized area measured from XT images in each experimental condition. The dash line represents the response induced by the flash only, after complete ER Ca 2+ store depletion. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. * P
    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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    Merck & Co thapsigargin
    Pathogenic GBA1 disrupts ER Ca 2+ release. (A–D) ER Ca 2+ release in GBA1 wt/wt 55 , GBA1 mut/mut 55 GD and GBA1 wt/mut 55 PD cells (young cohort). (A) Cytosolic Ca 2+ recordings from individual fibroblasts challenged with <t>thapsigargin</t> (1 μM) from the indicated representative populations. Experiments were performed in the absence of extracellular Ca 2+ . (B) Summary data (mean ± SEM) quantifying the magnitude of thapsigargin-evoked Ca 2+ signals in the indicated number of fields of view. Results are from 5 to 9 independent passages analysing 154–367 cells. (C) Cytosolic Ca 2+ recordings from individual fibroblasts stimulated with cADPR-AM (25 μM). Experiments were performed in the presence of extracellular Ca 2+ . (D) Summary data quantifying the percentage of cells responsive to cADPR. Results are from 2 to 3 independent passages analysing 39–75 cells. (E) Similar to A except thapsigargin-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (F) Summary data from 4 independent passages analysing 46–127 cells. (G) Similar to C except cADPR-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (H) Summary data from 3 to 6 independent passages analysing 73–257 cells. * p
    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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    92
    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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    94
    Cell Signaling Technology Inc thapsigargin
    VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with <t>thapsigargin</t> (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).
    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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    92
    Merck KGaA thapsigargin
    Fis1 expression leads to increased cytosolic calcium. ( A ) Cells were transfected and the cytosolic calcium concentration was measured after 27 h (Day 1) and 48 h (Day 2) after transfection by FACS using the Fluo-4/AM dye. The mean Fluo-4/AM fluorescent intensities of the β-gal-transfected cells were set to 100% in both Day 1 and Day 2, and the relative cytosolic calcium increase was calculated for Fis1- and p20Bap31-transfected cells. ( B ) The ER calcium content was measured in the same samples as A at Day 1 and Day 2 after transfection as the Ca 2+ released from the ER into the cytosol by <t>thapsigargin.</t> The ER calcium content was measured as the difference between the peak after addition of 10 μM thapsigargin (Tg) and the baseline fluorescence measured for 30 s (right panel). Similar to A , the mean ER calcium content measured by β-gal transfections was set to 100% on each day. ( C ) Cells were transfected with Fis1, with or without addition of zVAD-fmk. Cytosolic calcium was measured 27 h after transfection using the Fluo4/AM dye. The data are presented as the percentage reduction of cytosolic calcium in Fis1-transfected cells (without zVAD-fmk addition). ( D ) Baseline cytosolic calcium concentration of cells incubated either in DMEM containing 1.8 or 0.1 mM calcium were analysed. ( E ) Cells were transfected with the indicated genes, either in a medium containing 1.8 or 0.1 mM calcium. The percentage of subG1 population was analysed at 48 h p.t. ( F ) Cells were transfected with the indicated genes in two medium formulations, and the cleavage of Bap31–EYFP was determined after 24 h (top) and 30 h (bottom). ( G ) Cells were transfected either with β-gal or Fis1 in DMEM with 1.8 mM (lanes 1 and 4), 0.1 mM calcium (lanes 2 and 5), or 1.8 mM calcium with 5 μM BAPTA-AM (lanes 3 and 6). Cells were harvested at 27 h p.t. and the endogenous Bap31 cleavage was analysed by immunoblotting.
    Thapsigargin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM thapsigargin tg
    MISSL relocalizes to ALG-2-positive compartments upon a calcium rise in living cells. A, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were starved of amino acids for 60 min, and then an amino acid mixture was added ( t = 0). Time-lapse images were captured before (− AA ) and after (+ AA ) amino acid supplementation. Representative images of GFP-MISSL and R-GECO1 before (− AA, t = − 153 s ) and after (+ AA, t = 159 s ) are shown. Bar, 10 μm. B, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in A are plotted. C, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were treated with <t>thapsigargin</t> ( TG ) at t = 0. Time-lapse images before and after TG treatment were captured. Representative images of GFP-MISSL and R-GECO1 before ( t = − 124 s ) and after ( t = 83 s and t = 343 s ) are shown. Insets show magnified images of the region indicated by white squares. Bars, 5 μm. D, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in C are plotted.
    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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    92
    Valiant thapsigargin
    The L827P mutant inhibits WT IRE1α in HAP1 and Kms11 cells. A. IRE1GFP L827P inhibits XBP1 splicing in response to <t>thapsigargin</t> in leukemic HAP1 cells. Parental HAP1 cells expressing IRE1GFP L827P in addition to the endogenous WT IRE1α were induced with the indicated concentrations of dox (μg/ml) for 16 hr. The cells were then treated with 0.2 μM Tg for 4 hr and XBP1 splicing was assessed by RT-PCR. B. L827P IRE1GFP inhibits XBP1 splicing in response to tunicamycin in leukemic HAP1 cells. The same experiment as in A was performed except the cells were treated with 4 μg/ml Tm for 4 hr. C. L827P IRE1GFP inhibits ER stress-induced XBP1 splicing in multiple myeloma Kms11 cells. Kms11 cells expressing WT or L827P IRE1GFP were induced with dox for 16 hr and treated with 0.5 μM Tg for 4 hr where indicated. RNA was extracted and XBP1 splicing was assessed by RT-PCR and quantified. D. L827P inhibits RIDD activity in response to ER stress in Kms11 cells. The same samples as in C were used to perform a qPCR to detect BLOC1S1 expression levels, normalized over the unaffected ribosomal gene Rpl19. E. Expression of L827P decreases endogenous WT IRE1α phosphorylation in response to ER stress. Parental HAP1 cells, with constitutive expression of WT IRE1α and inducible expression of IRE1GFP L827P were induced with dox as above and then treated with Tg (0.2 μM for 4 hr). Cells were lysed and proteins were analyzed by western blot. Arrowhead: endogenous phospho-S724 IRE1α; *: non-specific bands. F. Quantification of phospho-IRE1α S724 and L827 mutant. The intensities of the western blot bands from the experiment described in E were normalized to total protein contents of the samples, measured by Ponceau S staining, quantified and plotted. G. L827P binds full-length WT IRE1α. HAP1KO cells were re-complemented with WT IRE1GFP, WT IRE1HA, D123P IRE1HA, L827P IRE1HA or combinations of constructs. The cells were induced with dox for 16 hr and treated with 4 μg/ml Tm for 4 hr where indicated. Cells were collected, lysed and subjected to immunoprecipitation with GFP-Trap beads. Beads-bound proteins were analyzed by western blot. Input: 5% of the lysates. Arrow: full length IRE1GFP or IRE1HA; §: lower molecular weight bands that appear to be IRE1α specific and size-sensitive to Tm treatment.
    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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    Extracellular rhHSP70 induces intracellular Ca 2+ mobilization A. rhHSP70 induced Ca 2+ release from internal stores in HMEC. Data are expressed as the 340/380 nm excitation ratio in one cell to observe oscillations in [Ca 2+ ]i because the oscillatory process is not synchronized in cells of the same monolayer. External additions of drugs are indicated by arrows. Changes in external calcium bath conditions are indicated on the bottom of traces. In most cases, drugs were initially applied in the absence (0 Ca) then in Ca 2+ (1.8 mM) containing solution to reveal Ca 2+ release from internal stores then external Ca 2+ entry, respectively (representative from 50 cells; n=10). No calcium increase was induced by the cell superfusion of the control bath solution (middle trace) while thapsigargin (TSG 4μM) always produced a drastic increase in [Ca 2+ ]i (lower trace; Representative from 50 cells; n=4). B. Contribution of EGFR to rhHSP70-induced Ca 2+ signaling. Superimposed traces from cells preincubated with the EGFR (ErbB1) inhibitor, gefitinib (10 μM for 30 min; in red), before the addition of rhHSP70 (Representative from 50 cells; n=5). C. Effects of phospholipase C (PLC) inhibitor U-73122 (5 μM) and its inactive analog U-73343 (5 μM) on the rhHSP70-induced Ca 2+ oscillations. Drugs were applied without (0 Ca) then with extracellular Ca 2+ (1.8 mM) (representative from 50 cells; n=5). D. Ca 2+ oscillations required both Ca 2+ release from internal stores and store operated Ca 2+ entry (SOCE). Cells were pretreated with the selective SOCE inhibitor, BTP-2 (20 μM; 20 min), before challenged with rhHSP70 (Representative from 30 cells; n=4).

    Journal: Oncotarget

    Article Title: Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells

    doi:

    Figure Lengend Snippet: Extracellular rhHSP70 induces intracellular Ca 2+ mobilization A. rhHSP70 induced Ca 2+ release from internal stores in HMEC. Data are expressed as the 340/380 nm excitation ratio in one cell to observe oscillations in [Ca 2+ ]i because the oscillatory process is not synchronized in cells of the same monolayer. External additions of drugs are indicated by arrows. Changes in external calcium bath conditions are indicated on the bottom of traces. In most cases, drugs were initially applied in the absence (0 Ca) then in Ca 2+ (1.8 mM) containing solution to reveal Ca 2+ release from internal stores then external Ca 2+ entry, respectively (representative from 50 cells; n=10). No calcium increase was induced by the cell superfusion of the control bath solution (middle trace) while thapsigargin (TSG 4μM) always produced a drastic increase in [Ca 2+ ]i (lower trace; Representative from 50 cells; n=4). B. Contribution of EGFR to rhHSP70-induced Ca 2+ signaling. Superimposed traces from cells preincubated with the EGFR (ErbB1) inhibitor, gefitinib (10 μM for 30 min; in red), before the addition of rhHSP70 (Representative from 50 cells; n=5). C. Effects of phospholipase C (PLC) inhibitor U-73122 (5 μM) and its inactive analog U-73343 (5 μM) on the rhHSP70-induced Ca 2+ oscillations. Drugs were applied without (0 Ca) then with extracellular Ca 2+ (1.8 mM) (representative from 50 cells; n=5). D. Ca 2+ oscillations required both Ca 2+ release from internal stores and store operated Ca 2+ entry (SOCE). Cells were pretreated with the selective SOCE inhibitor, BTP-2 (20 μM; 20 min), before challenged with rhHSP70 (Representative from 30 cells; n=4).

    Article Snippet: DiL-C18, thapsigargin and fura-2/AM were from Molecular Probes.

    Techniques: Produced, Planar Chromatography

    Subcellular location of ryanodine receptor, thapsigargin-sensitive Ca 2+ -ATPase, and endomembranes in mouse granulosa cells . The first block shows the intracellular location of the ryanodine receptor visualized by BODIPY FL-ryanodine. The second block depicts the intracellular location of thapsigargin-sensitive Ca 2+ -ATPase using BODIPY-FL thapsigargin. The third block shows the distribution of endomembranes by means of the BODIPY-FL conjugate of brefeldin A. All fluorescent probes were assayed at 1 μM (Yellow color). The second column shows the signal of the nuclear marker DAPI (Cyan color), whereas the third column depicts the merging the first and second columns. The fourth column is a magnification of a selected sector of the merge image. The results strongly suggest the intranuclear presence of all the Ca 2+ -handling proteins and the endomembranes in the mouse granulosa cells. Scale bar corresponds to 20 μm. Representative of 5 independent experiments.

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization

    doi: 10.1186/1477-7827-6-60

    Figure Lengend Snippet: Subcellular location of ryanodine receptor, thapsigargin-sensitive Ca 2+ -ATPase, and endomembranes in mouse granulosa cells . The first block shows the intracellular location of the ryanodine receptor visualized by BODIPY FL-ryanodine. The second block depicts the intracellular location of thapsigargin-sensitive Ca 2+ -ATPase using BODIPY-FL thapsigargin. The third block shows the distribution of endomembranes by means of the BODIPY-FL conjugate of brefeldin A. All fluorescent probes were assayed at 1 μM (Yellow color). The second column shows the signal of the nuclear marker DAPI (Cyan color), whereas the third column depicts the merging the first and second columns. The fourth column is a magnification of a selected sector of the merge image. The results strongly suggest the intranuclear presence of all the Ca 2+ -handling proteins and the endomembranes in the mouse granulosa cells. Scale bar corresponds to 20 μm. Representative of 5 independent experiments.

    Article Snippet: Fluo-4 AM, BODIPY TR-X Ryanodine, BODIPY-FL thapsigargin, TO-PRO-1 Iodide, brefeldin A BODIPY 558/568 conjugate isomer 1 were obtained from Molecular Probes (Eugene, OR.

    Techniques: Blocking Assay, Marker

    Effect of glucose on Ca 2+ leak and Ca 2+ extrusion in A7r5 VSM cells. A7r5 cells, cultured under HG, NG, and OC, were loaded with Fura2-AM and passive leak was monitored after store depletion with 1 μ M thapsigargin (TG), an irreversible SERCA inhibitor (a). Peak amplitude in response to TG (TG max release) and the time to maximum release are shown (b, c). Ca 2+ extrusion, which is presumed to be due to the combined activity of PMCA and NCX, was monitored as the decay of the TG-induced Ca 2+ transient in Ca 2+ free conditions to avoid Ca 2+ influx. To inhibit NCX, sodium was replaced with equimolar concentration of N-methyl-D-glucamine (d-e). Time to half ( T 1/2 ) decay was then measured and compared between the different conditions (d-e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Journal: BioMed Research International

    Article Title: Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling

    doi: 10.1155/2017/3691349

    Figure Lengend Snippet: Effect of glucose on Ca 2+ leak and Ca 2+ extrusion in A7r5 VSM cells. A7r5 cells, cultured under HG, NG, and OC, were loaded with Fura2-AM and passive leak was monitored after store depletion with 1 μ M thapsigargin (TG), an irreversible SERCA inhibitor (a). Peak amplitude in response to TG (TG max release) and the time to maximum release are shown (b, c). Ca 2+ extrusion, which is presumed to be due to the combined activity of PMCA and NCX, was monitored as the decay of the TG-induced Ca 2+ transient in Ca 2+ free conditions to avoid Ca 2+ influx. To inhibit NCX, sodium was replaced with equimolar concentration of N-methyl-D-glucamine (d-e). Time to half ( T 1/2 ) decay was then measured and compared between the different conditions (d-e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Article Snippet: Thapsigargin (TG) was from Invitrogen and ionomycin was from Life Technologies.

    Techniques: Cell Culture, Activity Assay, Concentration Assay

    Ca 2+ signaling pathways in normal (NHVSMC) and diabetic (DHVSMC) human aortic VSM cells. Cells were loaded with 2 μ M Fura-2AM for 30 min and passive leak was monitored after store depletion with 1 μ M thapsigargin (TG). Peak amplitude in response to TG (TG max release) and the time to half maximum release were compared between normal and diabetic cells (a, b). For the extrusion pathways the activity of PMCA and NCX was monitored by Ca 2+ imaging after store depletion with 1 μ M TG. Time to half ( T 1/2 ) decay was then measured and compared between the different conditions (c). Cells were labeled with 2 μ M Fura-2AM for 30 min and Ca 2+ release was imaged following treatment of the cells with 30 and 40 μ M of PE (d) or by using 30 μ M PE followed by 1 mM lanthanum chloride (La 3+ ) (e). Time to half decay was then measured and compared between normal and diabetics cells (e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Journal: BioMed Research International

    Article Title: Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling

    doi: 10.1155/2017/3691349

    Figure Lengend Snippet: Ca 2+ signaling pathways in normal (NHVSMC) and diabetic (DHVSMC) human aortic VSM cells. Cells were loaded with 2 μ M Fura-2AM for 30 min and passive leak was monitored after store depletion with 1 μ M thapsigargin (TG). Peak amplitude in response to TG (TG max release) and the time to half maximum release were compared between normal and diabetic cells (a, b). For the extrusion pathways the activity of PMCA and NCX was monitored by Ca 2+ imaging after store depletion with 1 μ M TG. Time to half ( T 1/2 ) decay was then measured and compared between the different conditions (c). Cells were labeled with 2 μ M Fura-2AM for 30 min and Ca 2+ release was imaged following treatment of the cells with 30 and 40 μ M of PE (d) or by using 30 μ M PE followed by 1 mM lanthanum chloride (La 3+ ) (e). Time to half decay was then measured and compared between normal and diabetics cells (e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Article Snippet: Thapsigargin (TG) was from Invitrogen and ionomycin was from Life Technologies.

    Techniques: Activity Assay, Imaging, Labeling

    Proliferation and Ca 2+ signaling in VSMC cells from normal (NHVSMC) and diabetic (DHVSMC) human aorta. For all the tests human normal and diabetic VSM cells were cultured under NG as recommended by the supplier. Cell proliferation was determined 24 and 48 h after plating, using the WST-1 assay (a). For Ca 2+ basal levels and Ca 2+ store content, cells were loaded with 2 μ M Fura-2AM for 30 min and the extent of Ca 2+ release was determined following treatment of the cells with 2 μ M ionomycin (b-c). For SOCE measurement, cells were loaded with 2 μ M Fura-2AM for 30 min and SOCE was determined after store depletion with 1 μ M thapsigargin (TG) (d). Cell contractility in response to 20 mM KCl was determined as described in Materials and Methods (e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Journal: BioMed Research International

    Article Title: Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling

    doi: 10.1155/2017/3691349

    Figure Lengend Snippet: Proliferation and Ca 2+ signaling in VSMC cells from normal (NHVSMC) and diabetic (DHVSMC) human aorta. For all the tests human normal and diabetic VSM cells were cultured under NG as recommended by the supplier. Cell proliferation was determined 24 and 48 h after plating, using the WST-1 assay (a). For Ca 2+ basal levels and Ca 2+ store content, cells were loaded with 2 μ M Fura-2AM for 30 min and the extent of Ca 2+ release was determined following treatment of the cells with 2 μ M ionomycin (b-c). For SOCE measurement, cells were loaded with 2 μ M Fura-2AM for 30 min and SOCE was determined after store depletion with 1 μ M thapsigargin (TG) (d). Cell contractility in response to 20 mM KCl was determined as described in Materials and Methods (e). Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Article Snippet: Thapsigargin (TG) was from Invitrogen and ionomycin was from Life Technologies.

    Techniques: Cell Culture, WST-1 Assay

    Effect of glucose Ca 2+ influx in A7r5 VSM cells. Effect of HG and NG on store-operated Ca 2+ entry (SOCE) (a, b) and on protein expression of STIM and Orai1 (c-d). Cells were loaded with Fura2-AM and SOCE stimulated after store depletion with 1 μ M thapsigargin (TG), an irreversible inhibitor of SERCA (a, b). Protein expression of STIM and Orai1 was determined between the different groups by western blot (c-d). Densitometry analysis was performed using Gene Tools, Geliance 600 Imaging system. Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Journal: BioMed Research International

    Article Title: Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling

    doi: 10.1155/2017/3691349

    Figure Lengend Snippet: Effect of glucose Ca 2+ influx in A7r5 VSM cells. Effect of HG and NG on store-operated Ca 2+ entry (SOCE) (a, b) and on protein expression of STIM and Orai1 (c-d). Cells were loaded with Fura2-AM and SOCE stimulated after store depletion with 1 μ M thapsigargin (TG), an irreversible inhibitor of SERCA (a, b). Protein expression of STIM and Orai1 was determined between the different groups by western blot (c-d). Densitometry analysis was performed using Gene Tools, Geliance 600 Imaging system. Data are presented as mean ± SE from at least three independent experiments done each in triplicate. ∗ p

    Article Snippet: Thapsigargin (TG) was from Invitrogen and ionomycin was from Life Technologies.

    Techniques: Expressing, Western Blot, Imaging

    Activation of ER stress by human oxidized LDL. (a) Western blotting analysis comparing changes in PERK, eIF2 and Ire1α and their phosphorylated forms (p). Total proteins were prepared from MIN6 cells cultured with 2 mmol/l cholesterol oxidized LDL (oxLDL) for the indicated times and 1 μmol/l thapsigargin (Thaps) for 6 h. The α-tubulin protein served as loading control. The figure is a representative experiment out of three. Measurement of CHOP / Chop , P58IPK / p58IPK and ATF4 / Atf4 mRNA levels in ( b) and (d) MIN6 and ( c) and (e) isolated human islets cells cultured with oxidized LDL. The mRNA level was quantified by quantitative real-time PCR in MIN6 or isolated human islets cells cultured for 48 h with vehicle (V), native (nLDL) or oxidized LDL (oxLDL). The PBA chemical chaperone 2.5 mmol/l were added in the cells cultured with oxidized LDL (oxLDL, filled bar ). For d ) and e ), cells were cultured with oxidized LDL plus 1 mmol/l cholesterol HDL ( filled bar ). The mRNA level was normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene ( RPLP0 / Rplp0 ) and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P

    Journal: PLoS ONE

    Article Title: Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL

    doi: 10.1371/journal.pone.0163046

    Figure Lengend Snippet: Activation of ER stress by human oxidized LDL. (a) Western blotting analysis comparing changes in PERK, eIF2 and Ire1α and their phosphorylated forms (p). Total proteins were prepared from MIN6 cells cultured with 2 mmol/l cholesterol oxidized LDL (oxLDL) for the indicated times and 1 μmol/l thapsigargin (Thaps) for 6 h. The α-tubulin protein served as loading control. The figure is a representative experiment out of three. Measurement of CHOP / Chop , P58IPK / p58IPK and ATF4 / Atf4 mRNA levels in ( b) and (d) MIN6 and ( c) and (e) isolated human islets cells cultured with oxidized LDL. The mRNA level was quantified by quantitative real-time PCR in MIN6 or isolated human islets cells cultured for 48 h with vehicle (V), native (nLDL) or oxidized LDL (oxLDL). The PBA chemical chaperone 2.5 mmol/l were added in the cells cultured with oxidized LDL (oxLDL, filled bar ). For d ) and e ), cells were cultured with oxidized LDL plus 1 mmol/l cholesterol HDL ( filled bar ). The mRNA level was normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene ( RPLP0 / Rplp0 ) and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P

    Article Snippet: Materials The 4-phenylbutyric acid (PBA) compound, N-acetylcystein (NAC) and thapsigargin were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activation Assay, Western Blot, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Expressing

    Fucoidan inhibits thapsigargin (Tg)-induced autophagy. A: RAW264.7 cells were left untreated or incubated with 0.5 μmol/L Tg or 0.5 μmol/L Tg plus 25 μg/mL fucoidan for 1, 2, 4, 8 or 12 hours. Cell lysates were subjected to Western blotting and detected by antibodies against LC3 and β-actin. B RAW264.7 cells were incubated for 12 hours with the indicated reagents, alone or in combination with 25 μg/mL fucoidan and 0.5 μmol/L Tg. Cell lysates were applied to Western blotting and detected by antibodies against LC3 and β-actin (left). The ratio of LC3-II to β-actin density was calculated using the ImageJ software and set to 1 for control (right). Non-treated cells were as control. Results were expressed as mean ± S.D. of triplicate samples. *P

    Journal: Journal of Biomedical Research

    Article Title: Class A scavenger receptor activation inhibits endoplasmic reticulum stress-induced autophagy in macrophage

    doi: 10.7555/JBR.28.20130105

    Figure Lengend Snippet: Fucoidan inhibits thapsigargin (Tg)-induced autophagy. A: RAW264.7 cells were left untreated or incubated with 0.5 μmol/L Tg or 0.5 μmol/L Tg plus 25 μg/mL fucoidan for 1, 2, 4, 8 or 12 hours. Cell lysates were subjected to Western blotting and detected by antibodies against LC3 and β-actin. B RAW264.7 cells were incubated for 12 hours with the indicated reagents, alone or in combination with 25 μg/mL fucoidan and 0.5 μmol/L Tg. Cell lysates were applied to Western blotting and detected by antibodies against LC3 and β-actin (left). The ratio of LC3-II to β-actin density was calculated using the ImageJ software and set to 1 for control (right). Non-treated cells were as control. Results were expressed as mean ± S.D. of triplicate samples. *P

    Article Snippet: Thapsigargin (Tg), 3-methyladenine (3-MA) and rapmycin were supplied by Sigma (St. Louis, MO, USA).

    Techniques: Incubation, Western Blot, Software

    Obesity-induced circulating factor converged on ER stress pathway to regulate TRIP-Br2 expression in vitro . qPCR analysis of TRIP-Br2 or ER stress marker (BiP) gene expression in ( a ) 3T3-L1 adipocytes after 24 h treatment with control (Ctrl) or compound indicated ( n =4 per group replicated twice); ( b ) 3T3-L1 adipocytes after 6, 24 or 48 h treatment with vehicle, thapsigargin (Thap; 0.1 μM) or tunicamycin (Tuni; 1 μg ml −1 ; n =3 per group replicated twice); ( c ) WT1 or C3H10T1/2 adipocytes after treatment with vehicle or Thap (0.1 μM) for 6, 24 or 48 h ( n =3 per group replicated twice); ( d ) 3T3-L1 preadipocytes treated with vehicle or Tuni (1 μg ml −1 ) for 24 h ( n =3 per group replicated twice); ( e ) human SGBS adipocytes treated with vehicle or Tuni (1 μg ml −1 ) for indicated time points ( n =3 per group replicated twice). ( f ) Western blot analysis for TRIP-Br2, BiP or β-actin (loading Ctrl) protein in 3T3-L1 adipocytes treated with vehicle (control, C) or Tuni (1 μg ml −1 , T) for indicated time points. ( g ) qPCR analysis of BiP gene expression in 3T3-L1 adipocytes treated with RAW- or HFD-fed gWAT-conditioned media for 24 h ( n =5 per group replicated twice) or 3T3-L1 adipocytes after 24 h treatment with Ctrl or compound indicated ( n =4 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P

    Journal: Nature Communications

    Article Title: The obesity-induced transcriptional regulator TRIP-Br2 mediates visceral fat endoplasmic reticulum stress-induced inflammation

    doi: 10.1038/ncomms11378

    Figure Lengend Snippet: Obesity-induced circulating factor converged on ER stress pathway to regulate TRIP-Br2 expression in vitro . qPCR analysis of TRIP-Br2 or ER stress marker (BiP) gene expression in ( a ) 3T3-L1 adipocytes after 24 h treatment with control (Ctrl) or compound indicated ( n =4 per group replicated twice); ( b ) 3T3-L1 adipocytes after 6, 24 or 48 h treatment with vehicle, thapsigargin (Thap; 0.1 μM) or tunicamycin (Tuni; 1 μg ml −1 ; n =3 per group replicated twice); ( c ) WT1 or C3H10T1/2 adipocytes after treatment with vehicle or Thap (0.1 μM) for 6, 24 or 48 h ( n =3 per group replicated twice); ( d ) 3T3-L1 preadipocytes treated with vehicle or Tuni (1 μg ml −1 ) for 24 h ( n =3 per group replicated twice); ( e ) human SGBS adipocytes treated with vehicle or Tuni (1 μg ml −1 ) for indicated time points ( n =3 per group replicated twice). ( f ) Western blot analysis for TRIP-Br2, BiP or β-actin (loading Ctrl) protein in 3T3-L1 adipocytes treated with vehicle (control, C) or Tuni (1 μg ml −1 , T) for indicated time points. ( g ) qPCR analysis of BiP gene expression in 3T3-L1 adipocytes treated with RAW- or HFD-fed gWAT-conditioned media for 24 h ( n =5 per group replicated twice) or 3T3-L1 adipocytes after 24 h treatment with Ctrl or compound indicated ( n =4 per group replicated twice). All qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean±s.e.m. Two-tailed Student's t -test or ANOVA, * P

    Article Snippet: Reagents Thapsigargin, tunicamycin (composition: A: 9.3%, B: 24.33%, C: 52.35%, D: 12.57%), TUDCA, LPS, palmitate, human insulin, isobutylmethylxanthine (IBMX), dexamethasone, T3, indomethacin, doxycycline, puromycin, G418, tamoxifen and polybrene were from Sigma-Aldrich.

    Techniques: Expressing, In Vitro, Real-time Polymerase Chain Reaction, Marker, Western Blot, Binding Assay, Two Tailed Test

    Inhibition of mitochondrial Hsp90s sensitizes HeLa cells toward thapsigargin. (A) Cytoplasmic calcium and mitochondrial membrane potential by suboptimal dose of gamitrinib. Fluo-4 or TMRM/MitoTracker-labeled HeLa cells were incubated with 5 μM gamitrinib for 24 hours and analyzed by confocal microscope. Bar, 20 μm. (B) Combination effect in HeLa. HeLa cells were treated with various concentrations of Thap in the presence of 5 μM of either 17AAG or gamitrinib, and analyzed by MTT assay (left). Alternatively, HeLa cells were treated with 5 μM gamitrinib and/or 0.06 μM Thap for 24 hours and analyzed by the MTT assay. ***, p

    Journal: Molecular Cancer

    Article Title: Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells

    doi: 10.1186/1476-4598-13-148

    Figure Lengend Snippet: Inhibition of mitochondrial Hsp90s sensitizes HeLa cells toward thapsigargin. (A) Cytoplasmic calcium and mitochondrial membrane potential by suboptimal dose of gamitrinib. Fluo-4 or TMRM/MitoTracker-labeled HeLa cells were incubated with 5 μM gamitrinib for 24 hours and analyzed by confocal microscope. Bar, 20 μm. (B) Combination effect in HeLa. HeLa cells were treated with various concentrations of Thap in the presence of 5 μM of either 17AAG or gamitrinib, and analyzed by MTT assay (left). Alternatively, HeLa cells were treated with 5 μM gamitrinib and/or 0.06 μM Thap for 24 hours and analyzed by the MTT assay. ***, p

    Article Snippet: 1,2-bis(o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA), cyclosporine A (CsA), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), tetracaine, and thapsigargin (Thap), and N-acetylcysteine (NAC) and all other chemicals, were from Sigma.

    Techniques: Inhibition, Labeling, Incubation, Microscopy, MTT Assay

    Localization of SelS and SelK and the translocation of p97(VCP). A and B , N2a cells were treated with 1 μg/ml Tm ( A ) or 100 n m thapsigargin ( Tg , B ) for 24 h and subjected to total cell extracts ( whole )/cytosol ( Cyto )/membrane ( Mem ) fraction analysis.

    Journal: The Journal of Biological Chemistry

    Article Title: Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation

    doi: 10.1074/jbc.M115.680215

    Figure Lengend Snippet: Localization of SelS and SelK and the translocation of p97(VCP). A and B , N2a cells were treated with 1 μg/ml Tm ( A ) or 100 n m thapsigargin ( Tg , B ) for 24 h and subjected to total cell extracts ( whole )/cytosol ( Cyto )/membrane ( Mem ) fraction analysis.

    Article Snippet: 24 h after seeding, the cells were treated with 1 μg/ml tunicamycin (Tm) and 100 n m thapsigargin or dimethyl sulfoxide (Sigma-Aldrich) ( , ) for 24 h.

    Techniques: Translocation Assay

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

    Journal: Frontiers in Cellular Neuroscience

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

    doi: 10.3389/fncel.2014.00016

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.18595

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

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

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

    C/EBPβ modulates EBV lytic gene expression and replication by ER stressors . EBV + Akata cells and Rael were treated with 20nM bortezomib (BZ), 1μM thapsigargin (TG), or 2 μg/mL tunicamycin (TU) for 24 hours. (A) RNA was isolated

    Journal: Blood

    Article Title: Bortezomib induction of C/EBP? mediates Epstein-Barr virus lytic activation in Burkitt lymphoma

    doi: 10.1182/blood-2011-01-332379

    Figure Lengend Snippet: C/EBPβ modulates EBV lytic gene expression and replication by ER stressors . EBV + Akata cells and Rael were treated with 20nM bortezomib (BZ), 1μM thapsigargin (TG), or 2 μg/mL tunicamycin (TU) for 24 hours. (A) RNA was isolated

    Article Snippet: Cells were treated for 24-48 hours with 20nM bortezomib (Millennium Pharmaceuticals), 1μM thapsigargin (Enzo Life Sciences), or 2 μg/mL tunicamycin (Enzo Life Sciences).

    Techniques: Expressing, Isolation

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

    Journal: Journal of Cell Science

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

    doi: 10.1242/jcs.116103

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

    Article Snippet: Thapsigargin was from Alomone Labs (Jerusalem, Israel).

    Techniques: Incubation

    Orlistat-induced ER stress causes synergism with gemcitabine (A) (B) Western blot analyses of ER stress-related protein levels in PANC-1 cells with indicated treatments at 48 hours post-treatment. (C) Isobologram showing the combination index of gemcitabine and thapsigargin in three different schedules: simultaneous, sequential, and reverse sequential in PANC-1. Combination index was calculated from MTT data using Compusyn software. (D) Relative percentage of PANC-1 cells dual positive for CD24/CD44 stem cell markers upon treatment with control, and thapsigargin for 48 hours. Gem: Gemcitabine, Thap: Thapsigargin. ** P ≤ 0.01, compared to the control by two sample Student’s t-test. (E) Kaplan-Meier survival comparisons between top and bottom tertiles for ER stress marker gene enrichment in TCGA patient tumor specimens. Comparisons are made for all stages (n = 20) and stage II only (n = 18) pancreatic ductal adenocarcinoma patients treated with gemcitabine only by utilizing Gehan-Breslow-Wilcoxon Test.

    Journal: Cancer research

    Article Title: De Novo Lipid Synthesis Facilitates Gemcitabine Resistance Through Endoplasmic Reticulum Stress in Pancreatic Cancer

    doi: 10.1158/0008-5472.CAN-16-3062

    Figure Lengend Snippet: Orlistat-induced ER stress causes synergism with gemcitabine (A) (B) Western blot analyses of ER stress-related protein levels in PANC-1 cells with indicated treatments at 48 hours post-treatment. (C) Isobologram showing the combination index of gemcitabine and thapsigargin in three different schedules: simultaneous, sequential, and reverse sequential in PANC-1. Combination index was calculated from MTT data using Compusyn software. (D) Relative percentage of PANC-1 cells dual positive for CD24/CD44 stem cell markers upon treatment with control, and thapsigargin for 48 hours. Gem: Gemcitabine, Thap: Thapsigargin. ** P ≤ 0.01, compared to the control by two sample Student’s t-test. (E) Kaplan-Meier survival comparisons between top and bottom tertiles for ER stress marker gene enrichment in TCGA patient tumor specimens. Comparisons are made for all stages (n = 20) and stage II only (n = 18) pancreatic ductal adenocarcinoma patients treated with gemcitabine only by utilizing Gehan-Breslow-Wilcoxon Test.

    Article Snippet: Orlistat, C75, Fatostatin, Thapsigargin (Cayman Chemical Company, Ann Arbor, MI, USA), and Platensimycin (Sigma-Aldrich Co., St. Louis, MO, USA) were dissolved in DMSO.

    Techniques: Western Blot, MTT Assay, Software, Marker

    PERK is required for B7H6 upregulation under ER stress conditions. B7H6 surface levels were assessed by flow cytometry after treatment with 0.125 μg/ml thapsigargin (Tg) or mock treated with DMSO for 16 h in the following conditions: a 624 wt, PERK knockout (KO), IRE1 KO and PERK/IRE1 double KO (DKO) cells, to the right appears quantification of the average mean fluorescence intensity (MFI) ± STD of treated relative to untreated cells of three independent experiments. b Melanoma 526 wt and PERK KO cells. c 624 wt cells pretreated with 1 μM GSK or 0.5 μM ISRIB for 1 h. The lower panel shows quantification of the average MFI ± STD of treated relative to untreated cells of three independent experiments. d 624 CHOP KO cells. BG indicates secondary only background staining, which was similar for both treated and untreated cells (shown is the BG for untreated cells)

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    Article Title: The integrated stress response promotes B7H6 expression

    doi: 10.1007/s00109-019-01859-w

    Figure Lengend Snippet: PERK is required for B7H6 upregulation under ER stress conditions. B7H6 surface levels were assessed by flow cytometry after treatment with 0.125 μg/ml thapsigargin (Tg) or mock treated with DMSO for 16 h in the following conditions: a 624 wt, PERK knockout (KO), IRE1 KO and PERK/IRE1 double KO (DKO) cells, to the right appears quantification of the average mean fluorescence intensity (MFI) ± STD of treated relative to untreated cells of three independent experiments. b Melanoma 526 wt and PERK KO cells. c 624 wt cells pretreated with 1 μM GSK or 0.5 μM ISRIB for 1 h. The lower panel shows quantification of the average MFI ± STD of treated relative to untreated cells of three independent experiments. d 624 CHOP KO cells. BG indicates secondary only background staining, which was similar for both treated and untreated cells (shown is the BG for untreated cells)

    Article Snippet: Chemicals and reagents Thapsigargin (Tg, abcam ab120286), GSK2606414 (GSK, TOCRIS 5107), cycloheximide (CHX, Sigma-Aldrich 66819), ISRIB (Sigma-Aldrich, SML0843), nelfinavir (Nel, Glentham Life Science, GP7332), lopinavir (Lop, Sigma-Aldrich, SML1222), ionomycin (Sigma-Aldrich, I3909), PMA (Sigma-Aldrich, P1585).

    Techniques: Flow Cytometry, Cytometry, Knock-Out, Fluorescence, Staining

    The effect of DLPhtEtn on PC-12 cell death induced by amyloid-β 1-40 peptide or thapsigargin . ( A ) PC-12 cells were treated with amyloid-β 1-40 peptide (5 μM) in the absence (Control) and presence of phospholipids (30 μM) as indicated in serum-free extracellular solution for 48 h, and cell viability was assayed. Data represents the mean (± SEM) percentage of basal levels (MTT intensities of cells untreated with amyloid-β 1-40 peptide) (n = 6 independent experiments). ** P

    Journal: Behavioral and Brain Functions : BBF

    Article Title: 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine ameliorates age-related spatial memory deterioration by preventing neuronal cell death

    doi: 10.1186/1744-9081-6-52

    Figure Lengend Snippet: The effect of DLPhtEtn on PC-12 cell death induced by amyloid-β 1-40 peptide or thapsigargin . ( A ) PC-12 cells were treated with amyloid-β 1-40 peptide (5 μM) in the absence (Control) and presence of phospholipids (30 μM) as indicated in serum-free extracellular solution for 48 h, and cell viability was assayed. Data represents the mean (± SEM) percentage of basal levels (MTT intensities of cells untreated with amyloid-β 1-40 peptide) (n = 6 independent experiments). ** P

    Article Snippet: Assay of cell viability PC-12 cells were treated with amyloid-β1-40 peptide (Peptide Institute Inc., Osaka, Japan) or thapsigargin (Wako, Osaka, Japan) in the presence and absence of phospholipids such as DLPhtEtn (Avanti Polar Lipid, Inc., Alabaster, AL, USA), 1-linoleoyl-2-palmitoyl-sn -glycero-3-phosphoethanolamine (LPPhtEtn) (Avanti Polar Lipid, Inc.), 1,2-dioleoyl-sn -glycero-3-phosphoethanolamine (DOPhtEtn) (Avanti Polar Lipid, Inc.), 1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine (DPPhtEtn) (Avanti Polar Lipid, Inc.), 1,2-diheptadecanoyl-sn -glycero-3-phosphoethanolamine (DHPhtEtn) (Avanti Polar Lipid, Inc.), 1,2-distearoyl -sn -glycero-3-phosphoethanolamine (DSPhtEtn) (Avanti Polar Lipid, Inc.), or DLPhtCho (Avanti Polar Lipid, Inc.), dissolved with PEG, and cell viability was evaluated by a dye staining method using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (DOJINDO, Kumamoto, Japan) by the method as previously described [ ].

    Techniques: MTT Assay

    Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM Thapsigargin or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

    doi: 10.1038/srep44113

    Figure Lengend Snippet: Multiple structurally distinct putative T-type (but not L-type) calcium channel blockers suppress Angiopoietin-2 (Angpt-2). 10 μM Flunarizine (FLU) or vehicle was applied for 24 hrs after pretreatment with ( A ) 10 μM Mibefradil or control for 1 h, ( B ) 50 μM TTA-A2 (a specific pharmacological inhibitor of t-type CCs) or control for 1 h, (C) 10 μM Amlodipine or control for 1 h. Angpt-2 concentration in the supernatant of human umbilical vein endothelial cells (HUVECs) was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6–10). (D) Before application of 10 μM FLU or vehicle for 24 hrs HUVECs were pretreated with 1 μM Thapsigargin or control for 0.5 hrs. Angpt-2 concentration in the supernatant was measured by ELISA and is shown relative to whole intracellular protein (n = 6–10). ( E ) 10 μM FLU or control was applied for 8 hrs after pretreatment with 10 μM EDTA for 1 h and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 6). Columns are presented as mean ± SEM.

    Article Snippet: 1 mM Wortmannin (Sigma-Aldrich, St. Louis, MO), 10 ng/ml Interleukin-1β (Life Technologies, Carlsbad, CA), 50 μM LY294002 (Cell Signaling Technology, Cambridge, UK), 100 ng/mL Phorbol-12-myristate-13-acetate (PMA) (Merck Millipore, Darmstadt, Germany), 10 μM Flunarizine dihydrochloride (Sigma-Aldrich, St. Louis, MO), 10 μM Amlodipine besylate (Sigma-Aldrich, St. Louis, MO), 10 μM Mibefradil dihydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), 50 μM TTA-A2 (Sigma-Aldrich, St. Louis, MO), 1 μM Thapsigargin (Santa Cruz Biotechnology, CA), 10 μM Ethylenediaminetetraacetic (Sigma-Aldrich, St. Louis, MO) and DMSO for molecular biology (Sigma-Aldrich, St. Louis, MO).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Pharmacology of IP 3 R response of local uncaging of caged Ca 2+ in CF15 cells in absence of extracellular Ca 2+ . A Example of line-scan images acquired at 2 ms per line and 0.21 μm per pixel in CF15 cells untreated at 37°C in presence or not of 100 μM 2-APB, 100 μM decavanadate, 20 mM caffeine or 10 μM cyclosporine A (all were preincubated during 10 min) and after 2 h incubation with 10 μM thapsigargin (TG). B Average of the line-scan images in A expressed as normalized fluorescence in each conditions C Mean normalized area measured from XT images in each experimental condition. The dash line represents the response induced by the flash only, after complete ER Ca 2+ store depletion. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. * P

    Journal: Respiratory Research

    Article Title: Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

    doi: 10.1186/1465-9921-9-70

    Figure Lengend Snippet: Pharmacology of IP 3 R response of local uncaging of caged Ca 2+ in CF15 cells in absence of extracellular Ca 2+ . A Example of line-scan images acquired at 2 ms per line and 0.21 μm per pixel in CF15 cells untreated at 37°C in presence or not of 100 μM 2-APB, 100 μM decavanadate, 20 mM caffeine or 10 μM cyclosporine A (all were preincubated during 10 min) and after 2 h incubation with 10 μM thapsigargin (TG). B Average of the line-scan images in A expressed as normalized fluorescence in each conditions C Mean normalized area measured from XT images in each experimental condition. The dash line represents the response induced by the flash only, after complete ER Ca 2+ store depletion. Results are presented as mean ± SEM and the number of experiments is noted on each bar graph. * P

    Article Snippet: Thapsigargin is from LC Laboratories.

    Techniques: Mass Spectrometry, Incubation, Fluorescence

    Pathogenic GBA1 disrupts ER Ca 2+ release. (A–D) ER Ca 2+ release in GBA1 wt/wt 55 , GBA1 mut/mut 55 GD and GBA1 wt/mut 55 PD cells (young cohort). (A) Cytosolic Ca 2+ recordings from individual fibroblasts challenged with thapsigargin (1 μM) from the indicated representative populations. Experiments were performed in the absence of extracellular Ca 2+ . (B) Summary data (mean ± SEM) quantifying the magnitude of thapsigargin-evoked Ca 2+ signals in the indicated number of fields of view. Results are from 5 to 9 independent passages analysing 154–367 cells. (C) Cytosolic Ca 2+ recordings from individual fibroblasts stimulated with cADPR-AM (25 μM). Experiments were performed in the presence of extracellular Ca 2+ . (D) Summary data quantifying the percentage of cells responsive to cADPR. Results are from 2 to 3 independent passages analysing 39–75 cells. (E) Similar to A except thapsigargin-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (F) Summary data from 4 independent passages analysing 46–127 cells. (G) Similar to C except cADPR-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (H) Summary data from 3 to 6 independent passages analysing 73–257 cells. * p

    Journal: Cell Calcium

    Article Title: Endoplasmic reticulum and lysosomal Ca2+ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts

    doi: 10.1016/j.ceca.2015.11.002

    Figure Lengend Snippet: Pathogenic GBA1 disrupts ER Ca 2+ release. (A–D) ER Ca 2+ release in GBA1 wt/wt 55 , GBA1 mut/mut 55 GD and GBA1 wt/mut 55 PD cells (young cohort). (A) Cytosolic Ca 2+ recordings from individual fibroblasts challenged with thapsigargin (1 μM) from the indicated representative populations. Experiments were performed in the absence of extracellular Ca 2+ . (B) Summary data (mean ± SEM) quantifying the magnitude of thapsigargin-evoked Ca 2+ signals in the indicated number of fields of view. Results are from 5 to 9 independent passages analysing 154–367 cells. (C) Cytosolic Ca 2+ recordings from individual fibroblasts stimulated with cADPR-AM (25 μM). Experiments were performed in the presence of extracellular Ca 2+ . (D) Summary data quantifying the percentage of cells responsive to cADPR. Results are from 2 to 3 independent passages analysing 39–75 cells. (E) Similar to A except thapsigargin-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (F) Summary data from 4 independent passages analysing 46–127 cells. (G) Similar to C except cADPR-evoked Ca 2+ release was assessed in GBA1 wt/wt 55 , GBA1 wt/mut 58 ASX and GBA1 wt/mut 55 PD cells. (H) Summary data from 3 to 6 independent passages analysing 73–257 cells. * p

    Article Snippet: Cells were stimulated with thapsigargin (Merck), cADPR-AM, synthesised as described previously and GPN (glycyl-l -phenylalanine 2-naphthylamide, SantaCruz Biotech).

    Techniques:

    ER Ca 2+ defects are age-dependent. (A) Cytosolic Ca 2+ recordings from individual fibroblasts challenged with thapsigargin (1 μM) from representative populations of GBA1 wt/wt 78 and GBA1 wt/mut 75 PD cells (aged cohort). (B) Summary data 3 independent passages analysing 112–117 cells. (C) Similar to A except, ER Ca 2+ release was assessed in GBA1 wt/wt 82 and GBA1 wt/mut 80 ASX cells. (D) Summary data from 3 independent passages analysing 131–134 cells. (E) ER Ca 2+ release from GBA1 wt/wt fibroblasts with increasing age. (F) Summary data from 1 to 14 independent passages analysing 30–483 cells. (G) Magnitude of ER Ca 2+ release versus age for both the young and aged cohort. All experiments were performed in the absence of extracellular Ca 2+ .

    Journal: Cell Calcium

    Article Title: Endoplasmic reticulum and lysosomal Ca2+ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts

    doi: 10.1016/j.ceca.2015.11.002

    Figure Lengend Snippet: ER Ca 2+ defects are age-dependent. (A) Cytosolic Ca 2+ recordings from individual fibroblasts challenged with thapsigargin (1 μM) from representative populations of GBA1 wt/wt 78 and GBA1 wt/mut 75 PD cells (aged cohort). (B) Summary data 3 independent passages analysing 112–117 cells. (C) Similar to A except, ER Ca 2+ release was assessed in GBA1 wt/wt 82 and GBA1 wt/mut 80 ASX cells. (D) Summary data from 3 independent passages analysing 131–134 cells. (E) ER Ca 2+ release from GBA1 wt/wt fibroblasts with increasing age. (F) Summary data from 1 to 14 independent passages analysing 30–483 cells. (G) Magnitude of ER Ca 2+ release versus age for both the young and aged cohort. All experiments were performed in the absence of extracellular Ca 2+ .

    Article Snippet: Cells were stimulated with thapsigargin (Merck), cADPR-AM, synthesised as described previously and GPN (glycyl-l -phenylalanine 2-naphthylamide, SantaCruz Biotech).

    Techniques:

    Inhibition of β-glucocerebrosidase does not affect ER Ca 2+ release. (A) Cytosolic Ca 2+ recordings from individual control GBA1 wt/wt fibroblasts challenged with thapsigargin (1 μM) from a representative population treated with 10 μM CBE for 8 days. (B) Summary data from 2 independent treatments analysing 87–90 cells. (C–F) Cytosolic Ca 2+ recordings from individual SH-SY5Y cells challenged with thapsigargin (1 μM) from a representative population treated with 10 μM CBE for 10–11 days (C) or stably expressing either scrambled shRNA ( GBA1 +/+ ) or shRNA targeting GBA1 ( GBA1 −/− ) (E). Summary data from 3 independent treatments analysing 117–204 cells (D) and 3 independent passages analysing 150–143 cells (F). All experiments were performed in the absence of extracellular Ca 2+ . ns, not significant. Inset (F) is a Western blot using antibodies to β-glucocerebrosidase (top) or actin (bottom) and homogenates (14 μg) from SH-SY5Y cells treated with the indicated shRNA.

    Journal: Cell Calcium

    Article Title: Endoplasmic reticulum and lysosomal Ca2+ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts

    doi: 10.1016/j.ceca.2015.11.002

    Figure Lengend Snippet: Inhibition of β-glucocerebrosidase does not affect ER Ca 2+ release. (A) Cytosolic Ca 2+ recordings from individual control GBA1 wt/wt fibroblasts challenged with thapsigargin (1 μM) from a representative population treated with 10 μM CBE for 8 days. (B) Summary data from 2 independent treatments analysing 87–90 cells. (C–F) Cytosolic Ca 2+ recordings from individual SH-SY5Y cells challenged with thapsigargin (1 μM) from a representative population treated with 10 μM CBE for 10–11 days (C) or stably expressing either scrambled shRNA ( GBA1 +/+ ) or shRNA targeting GBA1 ( GBA1 −/− ) (E). Summary data from 3 independent treatments analysing 117–204 cells (D) and 3 independent passages analysing 150–143 cells (F). All experiments were performed in the absence of extracellular Ca 2+ . ns, not significant. Inset (F) is a Western blot using antibodies to β-glucocerebrosidase (top) or actin (bottom) and homogenates (14 μg) from SH-SY5Y cells treated with the indicated shRNA.

    Article Snippet: Cells were stimulated with thapsigargin (Merck), cADPR-AM, synthesised as described previously and GPN (glycyl-l -phenylalanine 2-naphthylamide, SantaCruz Biotech).

    Techniques: Inhibition, Stable Transfection, Expressing, shRNA, Western Blot

    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

    VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with thapsigargin (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with thapsigargin (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    ATF6 has an active role in ER stress-induced VEGFA upregulation. ( A ) HepG2 cells were transfected with scramble (Control) and ATF6 siRNA and treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA and ATF6 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( B ) Luciferase activity in 293T cells transfected with VEGFA-promoter reporter (pGL3/VEGF −1039/0 ) or control pGL3 mock constructs along with indicated proteins and control pHA vector (values are mean ± SD). ATF6(Δ373) represents cleaved constitutive active form of ATF6 transcription factor. Xbp1-p and ATF5 represent positive and negative controls respectively. ( C ) Luciferase activity in 293T cells transfected with mouse VEGFA-intron 1 reporter (pGL3/VEGF 0/+3351 ) or control pGL3 mock constructs along with indicated proteins and control pHA vector (values are mean ± SD). ATF4 and ATF5 serve as positive and negative controls respectively.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: ATF6 has an active role in ER stress-induced VEGFA upregulation. ( A ) HepG2 cells were transfected with scramble (Control) and ATF6 siRNA and treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA and ATF6 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( B ) Luciferase activity in 293T cells transfected with VEGFA-promoter reporter (pGL3/VEGF −1039/0 ) or control pGL3 mock constructs along with indicated proteins and control pHA vector (values are mean ± SD). ATF6(Δ373) represents cleaved constitutive active form of ATF6 transcription factor. Xbp1-p and ATF5 represent positive and negative controls respectively. ( C ) Luciferase activity in 293T cells transfected with mouse VEGFA-intron 1 reporter (pGL3/VEGF 0/+3351 ) or control pGL3 mock constructs along with indicated proteins and control pHA vector (values are mean ± SD). ATF4 and ATF5 serve as positive and negative controls respectively.

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Construct, Plasmid Preparation

    Ire1α−/− mouse labyrinthine placenta defect is due to Ire1-dependent VEGFA expression. ( A ) WT and Ire1α−/− placenta were collected at d10.5 and vertically dissected and HE stained to show all placenta layers from maternal decidua to fetal chorionic plate SP = Spongiotrophoblast, LA = Labyrinthine trophoblast. ( B ) WT and Ire1α−/− embryo littermates were collected from Ire1α+/− females mated with Ire1α+/− males. Ire1α−/− animals die by embryonic day 10.5. ( C ) Labyrinthine trophoblast SM10 cells were treated with thapsigargin (Tg, 1 µM) tunicamycin (Tm, 5 µg/ml) or 2-Deoxy-glucose (2 DG) for 6 hrs. Total cell lysates and mRNA were collected. Lysates were analyzed by anti-Xbp1 showing the spliced active 55KD band, and anti-CHOP antibody (left panel). Total mRNA was used for expression analysis of VEGFA (right panel) (n = 2, values are mean ± SD). ( D ) SM10 cells were transfected with scramble (Control) or Hif1α siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and expression levels of HIF1α and VEGFA were measured by quantitative PCR (n = 3, values are mean ± SD). ( E ) SM10 cells were stably transduced with either LV/control GFP or LV/Ire1KA mutant lentivirus to constitutively express GFP or Ire1KA protein. Transduced stably expressing cells were kept in media with or without glucose for 24 hrs. Total mRNA was then analyzed for VEGFA, spliced XBP1 and CHOP expression (n = 2, values are mean ± SD).

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: Ire1α−/− mouse labyrinthine placenta defect is due to Ire1-dependent VEGFA expression. ( A ) WT and Ire1α−/− placenta were collected at d10.5 and vertically dissected and HE stained to show all placenta layers from maternal decidua to fetal chorionic plate SP = Spongiotrophoblast, LA = Labyrinthine trophoblast. ( B ) WT and Ire1α−/− embryo littermates were collected from Ire1α+/− females mated with Ire1α+/− males. Ire1α−/− animals die by embryonic day 10.5. ( C ) Labyrinthine trophoblast SM10 cells were treated with thapsigargin (Tg, 1 µM) tunicamycin (Tm, 5 µg/ml) or 2-Deoxy-glucose (2 DG) for 6 hrs. Total cell lysates and mRNA were collected. Lysates were analyzed by anti-Xbp1 showing the spliced active 55KD band, and anti-CHOP antibody (left panel). Total mRNA was used for expression analysis of VEGFA (right panel) (n = 2, values are mean ± SD). ( D ) SM10 cells were transfected with scramble (Control) or Hif1α siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and expression levels of HIF1α and VEGFA were measured by quantitative PCR (n = 3, values are mean ± SD). ( E ) SM10 cells were stably transduced with either LV/control GFP or LV/Ire1KA mutant lentivirus to constitutively express GFP or Ire1KA protein. Transduced stably expressing cells were kept in media with or without glucose for 24 hrs. Total mRNA was then analyzed for VEGFA, spliced XBP1 and CHOP expression (n = 2, values are mean ± SD).

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Expressing, Staining, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Mutagenesis

    Hif1α is not required for VEGFA induction under ER stress. ( A ) HepG2 cells were treated with either thapsigargin (Tg, 1 µM) 5 hr or hypoxia (0.5% O 2 ) for 24 hrs. Nuclear lysates were extracted and analyzed using anti-Hif1α and anti-Xbp1 antibodies. Spliced Xbp1 (55 kD) band is shown here. ( B ) HepG2 cells were transfected with scramble (Control) or Hif1α siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and expression levels of Hif1α and VEGFA were measured by quantitative PCR (n = 3, values are mean ± SD).

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: Hif1α is not required for VEGFA induction under ER stress. ( A ) HepG2 cells were treated with either thapsigargin (Tg, 1 µM) 5 hr or hypoxia (0.5% O 2 ) for 24 hrs. Nuclear lysates were extracted and analyzed using anti-Hif1α and anti-Xbp1 antibodies. Spliced Xbp1 (55 kD) band is shown here. ( B ) HepG2 cells were transfected with scramble (Control) or Hif1α siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and expression levels of Hif1α and VEGFA were measured by quantitative PCR (n = 3, values are mean ± SD).

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction

    ER stress induced VEGFA upregulation is also dependent on Perk. ( A ) WT and Perk−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Total mRNA was collected and VEGFA expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( B ) Perk−/− MEFs were stably transduced with LV/Perk, a lentivirus constitutively expressing Perk. These cells along with WT and Perk−/− MEFs and treated with thapsigargin (Tg, 1 µM) for 5 hrs. Cytoplasmic protein lysates were extracted and analyzed by immunoblot using anti-Perk antibody. Nuclear protein lysates were analyzed by immunoblot using anti-ATF4 and anti-Chop antibodies to show rescue of Perk signaling pathway. ( C )Total mRNA from (B) was analysed for VEGFA and ATF4 expression levels (n = 3, values are mean ± SD). ( D ) WT, Perk−/− and Perk−/− rescued MEFs were kept in media with or without glucose for 0, 24 and 48 hrs. Supernatant medium as well as whole cell lysates were collected and analyzed for VEGF protein concentration by ELISA. ( E ) ATF4−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and VEGFA expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( F ) HepG2 cells were transfected with scramble (Control) and Perk siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA, PERK and ATF4 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( G ) HepG2 cells were transfected with scramble (Control) and ATF4 siRNA and treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA and ATF4 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( H ) Luciferase activity in 293T cells transfected with mouse VEGFA-intron 1 reporter (pGL3/VEGF 0/+3351 ) or control pGL3 mock constructs along with indicated proteins and control PCDNA3 vector (values are mean ± SD). Bottom diagram shows schematic of mouse VEGFA gene as well as size of mouse VEGFA- intron construct. ( I ) Chromatin IP in Mouse neuro2a cells treated with or without thapsigargin (Tg, 1 µM) for 6 hrs was analyzed using quantitative PCR. PCR primers corresponded to mouse VEGFA-intron 1 genomic region. Fold enrichment was quantified using ratio of amplification of PCR product relative to 10% input DNA. Value obtained from mock was defined as 1(n = 3, values are mean ± SD).

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: ER stress induced VEGFA upregulation is also dependent on Perk. ( A ) WT and Perk−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Total mRNA was collected and VEGFA expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( B ) Perk−/− MEFs were stably transduced with LV/Perk, a lentivirus constitutively expressing Perk. These cells along with WT and Perk−/− MEFs and treated with thapsigargin (Tg, 1 µM) for 5 hrs. Cytoplasmic protein lysates were extracted and analyzed by immunoblot using anti-Perk antibody. Nuclear protein lysates were analyzed by immunoblot using anti-ATF4 and anti-Chop antibodies to show rescue of Perk signaling pathway. ( C )Total mRNA from (B) was analysed for VEGFA and ATF4 expression levels (n = 3, values are mean ± SD). ( D ) WT, Perk−/− and Perk−/− rescued MEFs were kept in media with or without glucose for 0, 24 and 48 hrs. Supernatant medium as well as whole cell lysates were collected and analyzed for VEGF protein concentration by ELISA. ( E ) ATF4−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 4 hrs. Total mRNA was collected and VEGFA expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( F ) HepG2 cells were transfected with scramble (Control) and Perk siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA, PERK and ATF4 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( G ) HepG2 cells were transfected with scramble (Control) and ATF4 siRNA and treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA and ATF4 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( H ) Luciferase activity in 293T cells transfected with mouse VEGFA-intron 1 reporter (pGL3/VEGF 0/+3351 ) or control pGL3 mock constructs along with indicated proteins and control PCDNA3 vector (values are mean ± SD). Bottom diagram shows schematic of mouse VEGFA gene as well as size of mouse VEGFA- intron construct. ( I ) Chromatin IP in Mouse neuro2a cells treated with or without thapsigargin (Tg, 1 µM) for 6 hrs was analyzed using quantitative PCR. PCR primers corresponded to mouse VEGFA-intron 1 genomic region. Fold enrichment was quantified using ratio of amplification of PCR product relative to 10% input DNA. Value obtained from mock was defined as 1(n = 3, values are mean ± SD).

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Protein Concentration, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Activity Assay, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    ER stress-induced VEGFA expression depends on Ire1. ( A–C ) WT and Ire1α−/− mouse embryonic fibroblasts (MEFs) were treated with ER stress inducers thapsigargin (Tg, 1 µM) 5 hr, tunicamycin (Tm, 5 µg/ml) 5 hr and hypoxia (0.5% O 2 ) for 24 hr. Total mRNA was collected and VEGFA and spliced Xbp1 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( D ) Ire1α−/− MEFs were stably transduced with LV/Ire1α or LV/GFP, a lentivirus constitutively expressing Ire1α or GFP. These cells along with WT and Ire1α−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Cytoplasmic lysates were analyzed using anti-Ire1α and anti-phosphorylated activated Ire1α (P-Ire1α) antibodies. Nuclear lysates were extracted and analyzed by anti-Xbp1 to show rescue of Ire1 signaling pathway (55 kD spliced form shown here) and anti-CHOP antibodies. ( E ) Total mRNA from (D) was analyzed for VEGFA and spliced Xbp1 expression levels (n = 3, values are mean ± SD). ( F ) WT, Ire1α−/− and Ire1α−/− rescued MEFs were kept in media with or without glucose for 24 hrs. Supernatant medium as well as whole cell lysates were collected and analyzed for VEGF protein concentration by ELISA. ( G ) Ire1α−/− MEFs transfected with processed-Xbp1 along with WT, Ire1α−/− and Ire1α−/− MEFs overexpressing Ire1α protein were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Total mRNA was analyzed for VEGFA and spliced Xbp1 expression levels (n = 3, values are mean ± SD). ( H ) HepG2 cells were transfected with scramble (Control), Ire1 siRNA or Xbp1 siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA, spliced Xbp1, IRE1α and EDEM expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( I ) Luciferase activity in 293T cells transfected with VEGFA-promoter reporter (pGL3/VEGF −1039/0 ) or control pGL3 mock constructs along with indicated proteins and control PCDNA3 vector (values are mean ± SD). Xbp1-p represents spliced constitutively active form of Xbp1 transcription factor. Bottom diagram shows schematic of mouse VEGFA gene as well as size of mouse VEGFA promoter construct. ( J ) Chromatin IP in Mouse neuro2a cells transfected with either mock pFlag-CMV-2 or processed-Xbp1 constructs and treated with or without thapsigargin (Tg, 1 µM) for 6 hrs was analyzed using quantitative PCR. PCR primer pair corresponded to mouse VEGFA promoter segment. Fold enrichment was quantified using ratio of amplification of PCR product relative to 10% input DNA. Value obtained from mock was defined as 1(n = 3, values are mean ± SD).

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

    doi: 10.1371/journal.pone.0009575

    Figure Lengend Snippet: ER stress-induced VEGFA expression depends on Ire1. ( A–C ) WT and Ire1α−/− mouse embryonic fibroblasts (MEFs) were treated with ER stress inducers thapsigargin (Tg, 1 µM) 5 hr, tunicamycin (Tm, 5 µg/ml) 5 hr and hypoxia (0.5% O 2 ) for 24 hr. Total mRNA was collected and VEGFA and spliced Xbp1 expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( D ) Ire1α−/− MEFs were stably transduced with LV/Ire1α or LV/GFP, a lentivirus constitutively expressing Ire1α or GFP. These cells along with WT and Ire1α−/− MEFs were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Cytoplasmic lysates were analyzed using anti-Ire1α and anti-phosphorylated activated Ire1α (P-Ire1α) antibodies. Nuclear lysates were extracted and analyzed by anti-Xbp1 to show rescue of Ire1 signaling pathway (55 kD spliced form shown here) and anti-CHOP antibodies. ( E ) Total mRNA from (D) was analyzed for VEGFA and spliced Xbp1 expression levels (n = 3, values are mean ± SD). ( F ) WT, Ire1α−/− and Ire1α−/− rescued MEFs were kept in media with or without glucose for 24 hrs. Supernatant medium as well as whole cell lysates were collected and analyzed for VEGF protein concentration by ELISA. ( G ) Ire1α−/− MEFs transfected with processed-Xbp1 along with WT, Ire1α−/− and Ire1α−/− MEFs overexpressing Ire1α protein were treated with thapsigargin (Tg, 1 µM) for 5 hrs. Total mRNA was analyzed for VEGFA and spliced Xbp1 expression levels (n = 3, values are mean ± SD). ( H ) HepG2 cells were transfected with scramble (Control), Ire1 siRNA or Xbp1 siRNA. 18 hrs post-transfection, cells were treated with thapsigargin (Tg, 1 µM) for 4 hr. Total mRNA was collected and VEGFA, spliced Xbp1, IRE1α and EDEM expression levels were measured by quantitative PCR (n = 3, values are mean ± SD). ( I ) Luciferase activity in 293T cells transfected with VEGFA-promoter reporter (pGL3/VEGF −1039/0 ) or control pGL3 mock constructs along with indicated proteins and control PCDNA3 vector (values are mean ± SD). Xbp1-p represents spliced constitutively active form of Xbp1 transcription factor. Bottom diagram shows schematic of mouse VEGFA gene as well as size of mouse VEGFA promoter construct. ( J ) Chromatin IP in Mouse neuro2a cells transfected with either mock pFlag-CMV-2 or processed-Xbp1 constructs and treated with or without thapsigargin (Tg, 1 µM) for 6 hrs was analyzed using quantitative PCR. PCR primer pair corresponded to mouse VEGFA promoter segment. Fold enrichment was quantified using ratio of amplification of PCR product relative to 10% input DNA. Value obtained from mock was defined as 1(n = 3, values are mean ± SD).

    Article Snippet: Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Protein Concentration, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Activity Assay, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Fis1 expression leads to increased cytosolic calcium. ( A ) Cells were transfected and the cytosolic calcium concentration was measured after 27 h (Day 1) and 48 h (Day 2) after transfection by FACS using the Fluo-4/AM dye. The mean Fluo-4/AM fluorescent intensities of the β-gal-transfected cells were set to 100% in both Day 1 and Day 2, and the relative cytosolic calcium increase was calculated for Fis1- and p20Bap31-transfected cells. ( B ) The ER calcium content was measured in the same samples as A at Day 1 and Day 2 after transfection as the Ca 2+ released from the ER into the cytosol by thapsigargin. The ER calcium content was measured as the difference between the peak after addition of 10 μM thapsigargin (Tg) and the baseline fluorescence measured for 30 s (right panel). Similar to A , the mean ER calcium content measured by β-gal transfections was set to 100% on each day. ( C ) Cells were transfected with Fis1, with or without addition of zVAD-fmk. Cytosolic calcium was measured 27 h after transfection using the Fluo4/AM dye. The data are presented as the percentage reduction of cytosolic calcium in Fis1-transfected cells (without zVAD-fmk addition). ( D ) Baseline cytosolic calcium concentration of cells incubated either in DMEM containing 1.8 or 0.1 mM calcium were analysed. ( E ) Cells were transfected with the indicated genes, either in a medium containing 1.8 or 0.1 mM calcium. The percentage of subG1 population was analysed at 48 h p.t. ( F ) Cells were transfected with the indicated genes in two medium formulations, and the cleavage of Bap31–EYFP was determined after 24 h (top) and 30 h (bottom). ( G ) Cells were transfected either with β-gal or Fis1 in DMEM with 1.8 mM (lanes 1 and 4), 0.1 mM calcium (lanes 2 and 5), or 1.8 mM calcium with 5 μM BAPTA-AM (lanes 3 and 6). Cells were harvested at 27 h p.t. and the endogenous Bap31 cleavage was analysed by immunoblotting.

    Journal: The EMBO Journal

    Article Title: Fis1 and Bap31 bridge the mitochondria-ER interface to establish a platform for apoptosis induction

    doi: 10.1038/emboj.2010.346

    Figure Lengend Snippet: Fis1 expression leads to increased cytosolic calcium. ( A ) Cells were transfected and the cytosolic calcium concentration was measured after 27 h (Day 1) and 48 h (Day 2) after transfection by FACS using the Fluo-4/AM dye. The mean Fluo-4/AM fluorescent intensities of the β-gal-transfected cells were set to 100% in both Day 1 and Day 2, and the relative cytosolic calcium increase was calculated for Fis1- and p20Bap31-transfected cells. ( B ) The ER calcium content was measured in the same samples as A at Day 1 and Day 2 after transfection as the Ca 2+ released from the ER into the cytosol by thapsigargin. The ER calcium content was measured as the difference between the peak after addition of 10 μM thapsigargin (Tg) and the baseline fluorescence measured for 30 s (right panel). Similar to A , the mean ER calcium content measured by β-gal transfections was set to 100% on each day. ( C ) Cells were transfected with Fis1, with or without addition of zVAD-fmk. Cytosolic calcium was measured 27 h after transfection using the Fluo4/AM dye. The data are presented as the percentage reduction of cytosolic calcium in Fis1-transfected cells (without zVAD-fmk addition). ( D ) Baseline cytosolic calcium concentration of cells incubated either in DMEM containing 1.8 or 0.1 mM calcium were analysed. ( E ) Cells were transfected with the indicated genes, either in a medium containing 1.8 or 0.1 mM calcium. The percentage of subG1 population was analysed at 48 h p.t. ( F ) Cells were transfected with the indicated genes in two medium formulations, and the cleavage of Bap31–EYFP was determined after 24 h (top) and 30 h (bottom). ( G ) Cells were transfected either with β-gal or Fis1 in DMEM with 1.8 mM (lanes 1 and 4), 0.1 mM calcium (lanes 2 and 5), or 1.8 mM calcium with 5 μM BAPTA-AM (lanes 3 and 6). Cells were harvested at 27 h p.t. and the endogenous Bap31 cleavage was analysed by immunoblotting.

    Article Snippet: To quantify the ER calcium content, 10 μM of thapsigargin (Merck Chemicals) was added to the tube after 30 s baseline calcium measurement.

    Techniques: Expressing, Transfection, Concentration Assay, FACS, Fluorescence, Incubation

    MISSL relocalizes to ALG-2-positive compartments upon a calcium rise in living cells. A, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were starved of amino acids for 60 min, and then an amino acid mixture was added ( t = 0). Time-lapse images were captured before (− AA ) and after (+ AA ) amino acid supplementation. Representative images of GFP-MISSL and R-GECO1 before (− AA, t = − 153 s ) and after (+ AA, t = 159 s ) are shown. Bar, 10 μm. B, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in A are plotted. C, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were treated with thapsigargin ( TG ) at t = 0. Time-lapse images before and after TG treatment were captured. Representative images of GFP-MISSL and R-GECO1 before ( t = − 124 s ) and after ( t = 83 s and t = 343 s ) are shown. Insets show magnified images of the region indicated by white squares. Bars, 5 μm. D, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in C are plotted.

    Journal: The Journal of Biological Chemistry

    Article Title: The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins

    doi: 10.1074/jbc.M117.800201

    Figure Lengend Snippet: MISSL relocalizes to ALG-2-positive compartments upon a calcium rise in living cells. A, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were starved of amino acids for 60 min, and then an amino acid mixture was added ( t = 0). Time-lapse images were captured before (− AA ) and after (+ AA ) amino acid supplementation. Representative images of GFP-MISSL and R-GECO1 before (− AA, t = − 153 s ) and after (+ AA, t = 159 s ) are shown. Bar, 10 μm. B, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in A are plotted. C, HeLa cells transiently expressing both GFP-MISSL and R-GECO1 were treated with thapsigargin ( TG ) at t = 0. Time-lapse images before and after TG treatment were captured. Representative images of GFP-MISSL and R-GECO1 before ( t = − 124 s ) and after ( t = 83 s and t = 343 s ) are shown. Insets show magnified images of the region indicated by white squares. Bars, 5 μm. D, changes of R-GECO1 fluorescent intensities in the area indicated by a rectangle in the R-GECO1 image in C are plotted.

    Article Snippet: Thapsigargin (TG) and brefeldin A (BFA) were purchased from Wako (Tokyo, Japan).

    Techniques: Expressing

    The L827P mutant inhibits WT IRE1α in HAP1 and Kms11 cells. A. IRE1GFP L827P inhibits XBP1 splicing in response to thapsigargin in leukemic HAP1 cells. Parental HAP1 cells expressing IRE1GFP L827P in addition to the endogenous WT IRE1α were induced with the indicated concentrations of dox (μg/ml) for 16 hr. The cells were then treated with 0.2 μM Tg for 4 hr and XBP1 splicing was assessed by RT-PCR. B. L827P IRE1GFP inhibits XBP1 splicing in response to tunicamycin in leukemic HAP1 cells. The same experiment as in A was performed except the cells were treated with 4 μg/ml Tm for 4 hr. C. L827P IRE1GFP inhibits ER stress-induced XBP1 splicing in multiple myeloma Kms11 cells. Kms11 cells expressing WT or L827P IRE1GFP were induced with dox for 16 hr and treated with 0.5 μM Tg for 4 hr where indicated. RNA was extracted and XBP1 splicing was assessed by RT-PCR and quantified. D. L827P inhibits RIDD activity in response to ER stress in Kms11 cells. The same samples as in C were used to perform a qPCR to detect BLOC1S1 expression levels, normalized over the unaffected ribosomal gene Rpl19. E. Expression of L827P decreases endogenous WT IRE1α phosphorylation in response to ER stress. Parental HAP1 cells, with constitutive expression of WT IRE1α and inducible expression of IRE1GFP L827P were induced with dox as above and then treated with Tg (0.2 μM for 4 hr). Cells were lysed and proteins were analyzed by western blot. Arrowhead: endogenous phospho-S724 IRE1α; *: non-specific bands. F. Quantification of phospho-IRE1α S724 and L827 mutant. The intensities of the western blot bands from the experiment described in E were normalized to total protein contents of the samples, measured by Ponceau S staining, quantified and plotted. G. L827P binds full-length WT IRE1α. HAP1KO cells were re-complemented with WT IRE1GFP, WT IRE1HA, D123P IRE1HA, L827P IRE1HA or combinations of constructs. The cells were induced with dox for 16 hr and treated with 4 μg/ml Tm for 4 hr where indicated. Cells were collected, lysed and subjected to immunoprecipitation with GFP-Trap beads. Beads-bound proteins were analyzed by western blot. Input: 5% of the lysates. Arrow: full length IRE1GFP or IRE1HA; §: lower molecular weight bands that appear to be IRE1α specific and size-sensitive to Tm treatment.

    Journal: bioRxiv

    Article Title: The interdomain helix between the kinase and RNase domains of IRE1α transmits the conformational change that underlies ER stress-induced activation

    doi: 10.1101/2020.01.14.902395

    Figure Lengend Snippet: The L827P mutant inhibits WT IRE1α in HAP1 and Kms11 cells. A. IRE1GFP L827P inhibits XBP1 splicing in response to thapsigargin in leukemic HAP1 cells. Parental HAP1 cells expressing IRE1GFP L827P in addition to the endogenous WT IRE1α were induced with the indicated concentrations of dox (μg/ml) for 16 hr. The cells were then treated with 0.2 μM Tg for 4 hr and XBP1 splicing was assessed by RT-PCR. B. L827P IRE1GFP inhibits XBP1 splicing in response to tunicamycin in leukemic HAP1 cells. The same experiment as in A was performed except the cells were treated with 4 μg/ml Tm for 4 hr. C. L827P IRE1GFP inhibits ER stress-induced XBP1 splicing in multiple myeloma Kms11 cells. Kms11 cells expressing WT or L827P IRE1GFP were induced with dox for 16 hr and treated with 0.5 μM Tg for 4 hr where indicated. RNA was extracted and XBP1 splicing was assessed by RT-PCR and quantified. D. L827P inhibits RIDD activity in response to ER stress in Kms11 cells. The same samples as in C were used to perform a qPCR to detect BLOC1S1 expression levels, normalized over the unaffected ribosomal gene Rpl19. E. Expression of L827P decreases endogenous WT IRE1α phosphorylation in response to ER stress. Parental HAP1 cells, with constitutive expression of WT IRE1α and inducible expression of IRE1GFP L827P were induced with dox as above and then treated with Tg (0.2 μM for 4 hr). Cells were lysed and proteins were analyzed by western blot. Arrowhead: endogenous phospho-S724 IRE1α; *: non-specific bands. F. Quantification of phospho-IRE1α S724 and L827 mutant. The intensities of the western blot bands from the experiment described in E were normalized to total protein contents of the samples, measured by Ponceau S staining, quantified and plotted. G. L827P binds full-length WT IRE1α. HAP1KO cells were re-complemented with WT IRE1GFP, WT IRE1HA, D123P IRE1HA, L827P IRE1HA or combinations of constructs. The cells were induced with dox for 16 hr and treated with 4 μg/ml Tm for 4 hr where indicated. Cells were collected, lysed and subjected to immunoprecipitation with GFP-Trap beads. Beads-bound proteins were analyzed by western blot. Input: 5% of the lysates. Arrow: full length IRE1GFP or IRE1HA; §: lower molecular weight bands that appear to be IRE1α specific and size-sensitive to Tm treatment.

    Article Snippet: Tunicamycin and 4μ8c were purchased from Calbiochem, thapsigargin was from MP Biomedicals and Luteolin was from Sigma.

    Techniques: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Staining, Construct, Immunoprecipitation, Molecular Weight

    IRE1α L827P is an enzymatically inactive mutant. A. Failure of IRE1α L827P to support the XBP1 splicing activity. HAP1KO cells were complemented by either the revertant wild type human IRE1GFP (P827L, see text) or the L827P mutant. Each stable subline was subjected to treatment with 4 μg/ml tunicamycin for the indicated times, after which splicing of XBP1 transcripts was assayed by a RT-PCR gel assay. Specificity of the assay for IRE1α activity was assessed by inclusion of 4μ8c (16 μM) where indicated. u: unspliced XBP1; s: spliced XBP1; *: hybrid band resulting from unspliced and spliced XBP1 (Li et al., 2010). B. IRE1GFP L827P cannot be activated by the flavonoid luteolin. HAP1 cells with WT or L827P IRE1α as in A, were treated with 0.2μM thapsigargin, or the IRE1α cytosolic activator luteolin (50 μM), or with the DMSO solvent alone (-) for 2 hr. XBP1 splicing was then assayed as in A. C. Failure of IRE1α L827P to support RIDD activity. The same stable sublines were stressed as in A and then the RIDD activity of IRE1α was assayed using qPCR quantitation of the common BLOC1S1 substrate. Data are expressed as the relative abundance of BLOC1S1 under each condition relative to the abundance of the unaffected ribosomal gene Rpl19. Values are means ± SEM of triplicate measurements in two independent experiments. D. IRE1GFP L827P does not cluster in response to ER stress. HAP1KO cells re-complemented with WT or L827P IRE1GFP were induced with dox for 16 hr. The following day they were treated with Tm (4 μg/ml) and imaged over 8 hr. Images are representative fields of the 4-hr treatment. E. Quantification of clustering. The images from Fig. 1D were quantified and plotted. F. L827P IRE1GFP is not phosphorylated on S729 following induction of ER stress. HAP1KO re-complemented with WT or L827P IRE1GFP were stressed with Tm as in D and with SubAB at the indicated concentrations for 2 hr. Cells were lysed and activation of IRE1α was assessed by western blot with an antibody against phosphorylated Ser729 of IRE1α. 14.3.3: housekeeping protein. Arrow indicates full length IRE1GFP; arrowhead: phospho-IRE1GFP S729; §: lower molecular weight bands that appear to be IRE1α specific and size-sensitive to Tm treatment; *: non-specific bands. G. Location of L827 in the crystal structure of IRE1α. Residue L827 of human IRE1α is highlighted in red in PDB 5HGI. For orientation, the catalytic residue in the kinase domain, K599, is marked in blue and the catalytic residue in the RNase domain, K907, is marked in purple.

    Journal: bioRxiv

    Article Title: The interdomain helix between the kinase and RNase domains of IRE1α transmits the conformational change that underlies ER stress-induced activation

    doi: 10.1101/2020.01.14.902395

    Figure Lengend Snippet: IRE1α L827P is an enzymatically inactive mutant. A. Failure of IRE1α L827P to support the XBP1 splicing activity. HAP1KO cells were complemented by either the revertant wild type human IRE1GFP (P827L, see text) or the L827P mutant. Each stable subline was subjected to treatment with 4 μg/ml tunicamycin for the indicated times, after which splicing of XBP1 transcripts was assayed by a RT-PCR gel assay. Specificity of the assay for IRE1α activity was assessed by inclusion of 4μ8c (16 μM) where indicated. u: unspliced XBP1; s: spliced XBP1; *: hybrid band resulting from unspliced and spliced XBP1 (Li et al., 2010). B. IRE1GFP L827P cannot be activated by the flavonoid luteolin. HAP1 cells with WT or L827P IRE1α as in A, were treated with 0.2μM thapsigargin, or the IRE1α cytosolic activator luteolin (50 μM), or with the DMSO solvent alone (-) for 2 hr. XBP1 splicing was then assayed as in A. C. Failure of IRE1α L827P to support RIDD activity. The same stable sublines were stressed as in A and then the RIDD activity of IRE1α was assayed using qPCR quantitation of the common BLOC1S1 substrate. Data are expressed as the relative abundance of BLOC1S1 under each condition relative to the abundance of the unaffected ribosomal gene Rpl19. Values are means ± SEM of triplicate measurements in two independent experiments. D. IRE1GFP L827P does not cluster in response to ER stress. HAP1KO cells re-complemented with WT or L827P IRE1GFP were induced with dox for 16 hr. The following day they were treated with Tm (4 μg/ml) and imaged over 8 hr. Images are representative fields of the 4-hr treatment. E. Quantification of clustering. The images from Fig. 1D were quantified and plotted. F. L827P IRE1GFP is not phosphorylated on S729 following induction of ER stress. HAP1KO re-complemented with WT or L827P IRE1GFP were stressed with Tm as in D and with SubAB at the indicated concentrations for 2 hr. Cells were lysed and activation of IRE1α was assessed by western blot with an antibody against phosphorylated Ser729 of IRE1α. 14.3.3: housekeeping protein. Arrow indicates full length IRE1GFP; arrowhead: phospho-IRE1GFP S729; §: lower molecular weight bands that appear to be IRE1α specific and size-sensitive to Tm treatment; *: non-specific bands. G. Location of L827 in the crystal structure of IRE1α. Residue L827 of human IRE1α is highlighted in red in PDB 5HGI. For orientation, the catalytic residue in the kinase domain, K599, is marked in blue and the catalytic residue in the RNase domain, K907, is marked in purple.

    Article Snippet: Tunicamycin and 4μ8c were purchased from Calbiochem, thapsigargin was from MP Biomedicals and Luteolin was from Sigma.

    Techniques: Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitation Assay, Activation Assay, Western Blot, Molecular Weight