thapsigargin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher thapsigargin
    Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with <t>BODIPY</t> FL <t>thapsigargin</t> (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P
    Thapsigargin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Thermo Fisher
    Average 99 stars, based on 804 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Millipore thapsigargin
    Effect of TNFα on proximity of mitochondria to the sarcoplasmic reticulum (SR). Top : fluorescence images of human ASM cells loaded simultaneously with <t>BODIPY</t> FL <t>thapsigargin</t> (endoplasmic reticulum Ca 2+ pumps, green) to visualize SR and MitoTracker Red CMXRos to visualize mitochondria (red). Bottom : Manders' colocalization coefficients M1 (mitochondria colocalization with the SR) and M2 (SR colocalization with mitochondria) were used to estimate proximity of mitochondria to the SR. In ASM cells exposed to TNFα, proximity of mitochondria to the SR ( M1 ) was significantly decreased. Values are means ± SE. *Significant effect ( P
    Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Millipore
    Average 99 stars, based on 6618 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Tocris thapsigargin
    Cytosolic Ca 2+ triggers cell proliferation and EE differentiation through different mechanisms. a,b. Increase of cytosolic Ca 2+ by channelrhodopsin (ChR) in Dl + and Piezo + EP cells. Dl + , Piezo + , and EE cell numbers are quantified. Number of areas quantified: n=28 (Dark, Dl-Gal4 ), n=30 (Light+ATR, Dl-Gal4 ), n=30 (Dark, Piezo-Gal4 ), n=31 (Light+ATR, Piezo-Gal4 ). c,d. Midguts of <t>Thapsigargin</t> (Thap) and Trametinib (Tram) treated flies. Number of areas quantified: n=29 (Ctl), n=31 (Thap), n=32 (Thap+Tram), n=29 (Tram). Data are expressed as mean + s.e.m. P-values are from two-tailed t-test. Scale bar, 50 μm.
    Thapsigargin, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Tocris
    Average 99 stars, based on 420 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    92
    Alomone Labs thapsigargin
    Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM <t>thapsigargin</t> in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P
    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
    https://www.bioz.com/result/thapsigargin/product/Alomone Labs
    Average 92 stars, based on 112 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    95
    Abcam thapsigargin
    Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM <t>thapsigargin</t> in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P
    Thapsigargin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Abcam
    Average 95 stars, based on 78 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    95/100 stars
      Buy from Supplier

    92
    Cayman Chemical thapsigargin
    Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM <t>Thapsigargin;</t> GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p
    Thapsigargin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Cayman Chemical
    Average 92 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    92
    FUJIFILM thapsigargin
    IFNα exerts direct cytotoxicity to HBsAg accumulating hepatocytes and downregulate UPR. (a-c) Effects of IFN signaling and UPR modulation on HBsAg accumulating hepatocytes in vitro. (A) Experimental design. (B) LDH levels in the supernatant of PMH culture were measured at indicated time points after adding medium (white bars) or IFNα (0.1MU/ml) (black bars). (C) The immunoblots of UPR related molecules and ISG15 at specified time points after IFNα treatment. (D, E) IFNα suppresses chemically induced UPR in vitro. (D) Experimental schema. (E) Representative immunoblots show the effect of IFNα on UPR related-protein levels at 0, 6, and 12 hours after treatment with <t>thapsigargin.</t>
    Thapsigargin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/FUJIFILM
    Average 92 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    92
    Enzo Biochem thapsigargin
    Peroxisome deficiency does not enhance ER-to-Golgi trafficking of GFP-ATF6(S1P-). Confocal microscopy images show GFP-ATF6(S1P-) trafficking related to the Golgi marker giantin in CHO-K1 and ZR-82 cells. Cells were either cultured in 10% FCS (Control) (A) or treated with 100 nM <t>thapsigargin</t> (B) or 0.5 μg/ml tunicamycin (C) for 12 h. GFP-ATF6 was visualized using a chicken polyclonal anti-GFP antibody; the Golgi was visualized by rabbit anti-giantin; DAPI was used for nuclear staining. Representative fields are shown for each condition. The scale bars represent 10 μm.
    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
    https://www.bioz.com/result/thapsigargin/product/Enzo Biochem
    Average 92 stars, based on 165 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    92
    LC Laboratories thapsigargin
    <t>Thapsigargin-dependent</t> Ca 2+ entry in S2 cells. Fluo-4 fluorescence changes were monitored using a FLIPR 384 . After 20 s of recording, the 384-well pipette-tip head was lowered into the solution creating an offset artifact in the recording. This offset artifact is unrelated to a cellular response and is dependent on the fluid volume in each well at the start of the experiment and the extent of tip penetration into the solution. 10 s after lowering the pipette-tip head, either thapsigargin (TG, 1 μM final, circles) or DMSO (triangles) was injected (arrow). CaCl 2 was then added to achieve a final concentration of 1.8 mM. Traces were zeroed at time 0, and each data point represents the mean (±SEM) of 192 replicates.
    Thapsigargin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/LC Laboratories
    Average 92 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc thapsigargin
    <t>Thapsigargin</t> could not mimic the effects of tunicamycin on MDR GC cells. a Concentration-survival curves of GC cells treated with Tg or Tu (wide dose range) for 48 h. Tg, thapsigargin. b Expressions of UPR-related proteins in GC cells after Tg treatment (2 μg/ml) for 48 h determined by WB. All proteins were normalized to β-actin. c The effects of Tg on the chemosensitivity of GC cells after treatment for 48 h. ns, non-significant; * P
    Thapsigargin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Cell Signaling Technology Inc
    Average 94 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    94/100 stars
      Buy from Supplier

    92
    Merck & Co thapsigargin
    Synergism between ER stress and other stimuli. a FLS ( n = 4) were left untreated or stimulated with 1 μg/ml LPS or 1 ng/ml IL-1β in the presence or absence of 10 nM TG. Expression of IL6 and IL8 was measured by qPCR 4 h or 8 h after stimulation. b , c FLS ( n = 3, n = 2 for TG alone) were stimulated with 10 nM TG or 1 μg/ml LPS in the presence or absence of 10 nM TG for 8 h. Expression of 84 inflammatory genes was monitored by qPCR-based array. Heat map ( b ) depicts per-gene z-scores of log-scaled expression values relative to GAPDH, and bar graph ( c ) presents fold changes relative to unstimulated cells for 30 genes with the highest overall level of regulation (mean across all conditions). ER Endoplasmic reticulum, FLS Fibroblast-like synoviocytes, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, IL Interleukin, LPS Lipopolysaccharide, qPCR Quantitative polymerase chain reaction, TG <t>Thapsigargin</t>
    Thapsigargin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Merck & Co
    Average 92 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/thapsigargin/product/Biomol GmbH
    Average 92 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    92
    Merck KGaA thapsigargin
    NAADP-elicited Ca 2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice ( n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice ( n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM <t>thapsigargin</t> ( n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca 2+ responses to NAADP (100 nM), IP 3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 ( n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.
    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
    https://www.bioz.com/result/thapsigargin/product/Merck KGaA
    Average 92 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    99
    Millipore reagents thapsigargin
    Flx affects osteoclastogenesis in a 5HTT-independent manner ( a ) Quantification of TRAP activity (left) and gene expression in primary osteoclast cultures (OCs) derived from 5HTT-deficient ( Slc6a4 −/− ) mice. Ctsk, Cathepsin K. Clcn7, Chloride channel 7. Acp5 encodes TRAP. ( b ) Representative images ( n = 4 images/mouse) of vertebrae (left) from Slc6a4 −/− females treated for 3 w (veh n = 8, Flx n = 6). Quantification of BV/TV is indicated below each image. Scale bars, 200 μm. Bone histomorphometry of these mice is also indicated (middle and right). ( c , d ) Gene expression in WT OCs ( c ) and Slc6a4 −/− OCs ( d ). Mitf encodes microphthalmia-associated transcription factor; Undiff., undifferentiated OCs. ( e ) Expression of Nfatc1 in long bones of WT females treated for 3 w. ( f ) Western blot (left) and quantification of band intensities reported to veh (right) of indicated proteins in RAW264.7 osteoclasts treated with Flx or veh ( n = 1 technical replicate of the number of biological replicates indicated in the bar graph (left)). ( g ) Representative curves ( n = 13) of Fura-2 ratio (340/380) in individual RAW264.7 osteoclasts recorded in veh medium and after addition of Flx (left), Fura-2 ratio levels after Flx addition relative to veh (AUC, area under the curve ( n = 13) (middle), and proportion of OCs and MNCs responding to Flx (right). <t>Thap,</t> <t>Thapsigargin.</t> ( h ) Gene expression in RAW264.7 osteoclasts treated for 24 h. ( i ) Gene expression in Creb fl/fl:virus-CRE OCs treated for 6 days (6 d) (left), and the percentage of resorbed area in a pit resorption assay of WT OCs treated for 24 h with W5, KG-501 and Flx (right). ( j ) Gene expression in spleen of WT or Slc6a4 −/− females treated for 3 weeks. Rorc encodes RORγt. Values are mean ± SEM compared to veh (*) or undiff. OCs ( $ ) */ $ P ≤ 0.05, ** P ≤ 0.01, ***/ $$$ P ≤ 0.001, ****/ $$$$ P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test ( a , i ), one-way ANOVA followed by Turkey’s ( c , d , h ) or Student’s test (all other panels).
    Reagents Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reagents thapsigargin/product/Millipore
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    reagents thapsigargin - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    92
    FUJIFILM thapsigargin tg
    Supplemental expression of PIMT reduces EMT and cell invasion in A549 cells induced by <t>Thapsigargin</t> ( A ) Immunoblotting of PIMT and E-cadherin in A549 cells treated with 0.1 µM of Tg, and PIMT expression and empty vectors. ( B, C ) Intensity of PIMT and E-cadherin in A549 cells treated with Tg, and PIMT expression and empty vectors. * indicates p
    Thapsigargin Tg, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin tg/product/FUJIFILM
    Average 92 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    thapsigargin tg - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/thapsigargin/product/Valiant
    Average 92 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    95
    Millipore 2 4 6 trihydroxyacetophenone monohydrate thap
    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.
    2 4 6 Trihydroxyacetophenone Monohydrate Thap, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/2 4 6 trihydroxyacetophenone monohydrate thap/product/Millipore
    Average 95 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    2 4 6 trihydroxyacetophenone monohydrate thap - by Bioz Stars, 2021-01
    95/100 stars
      Buy from Supplier

    92
    Thermo Fisher thapsigargin tg
    ER stress was activated by TG and DTT in HUVECs. Relative mRNA expression by qPCR of ER stress markers: BiP (A) , CHOP (B) , and TRIB3 (C) in HUVECs treated with <t>thapsigargin</t> (TG; 300 nM, 24 h) and BiP (D) , CHOP (E) , ATF-4 (F) and TRIB3 (G) in HUVECs treated with Dithiothreitol (DTT; 2M, 24 h) normalized against housekeeping gene β -actin ( n = 3–4). (H) , Western blot of protein expression of BiP in HUVECs treated with thapsigargin (TG; 300 nM, 24 h) in the presence or absence of PBA (10 mM). Bars represent pooled densitometry data normalized to total amount of β-actin loading control expressed as fold change compared to control (CTL) ( n = 4 per group). Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA followed with Tukey's multiple comparison test. ** P
    Thapsigargin Tg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin tg/product/Thermo Fisher
    Average 92 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    thapsigargin tg - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    N/A
    In cells sarco endoplasmic calcium ATPases SERCAs transport free calcium into the sarcoplasmic and endoplasmic reticula lowering intracellular calcium levels to stop signaling through this cation Thapsigargin is a non
      Buy from Supplier


    N/A
    Thapsigargin is a cell permeable sesquiterpene lactone found in the roots of Thapsia garganica Thapsigargin is a tumor promoter and induces the release of intracellular stored Ca2 without hydrolysis of
      Buy from Supplier

    N/A
    Potent cell permeable IP3 independent intracellular calcium releaser Inhibits microsomal Ca2 ATPase Stimulates arachidonic acid metabolism in macrophages
      Buy from Supplier

    Image Search Results


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

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

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

    doi: 10.1152/ajplung.00538.2016

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

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

    Techniques: Fluorescence

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

    Journal: Nature

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

    doi: 10.1038/nature25744

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

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

    Techniques: CTL Assay, Two Tailed Test

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

    Journal: Nature

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

    doi: 10.1038/nature25744

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

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

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

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

    Journal: Experimental eye research

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

    doi: 10.1016/j.exer.2017.01.002

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

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

    Techniques: Real-time Polymerase Chain Reaction

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: Expressing, Fluorescence, Translocation Assay

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: Transfection, Expressing, Fluorescence

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: Transfection, Plasmid Preparation

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

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

    Article Snippet: Thapsigargin was from Alomone Labs.

    Techniques: Inhibition, Transfection, Fluorescence

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201506022

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

    Article Snippet: Thapsigargin was from Alomone Labs.

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

    Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

    doi: 10.1186/s12958-020-00643-7

    Figure Lengend Snippet: Inhibitor effects on on long-lasting [Ca 2+ ] i oscillations. a Cytoplasmic [Ca 2+ ] i dynamic changes after inhibitor addition. b Development of inhibitor addition following [Ca 2+ ] i oscillations initiation. PA indicates survival of embryos of all MII oocytes. PN indicates pronuclear embryos of surviving oocytes. 2-Cell indicates cleaved embryos of pronuclear embryos. RR: 10 μM Ruthenium Red; Tha: 1 μM Thapsigargin; GSK: 1 mM GSK-7975A; Mib: Mibefradil; N: 1 μM NS-8593. The significance of differences between groups was analyzed by the Chi-square test and p

    Article Snippet: The details of Ca2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

    Techniques:

    Effect of SERCAs inhibitor Thapsigargin on oocyte activation. a Cytoplasmic ([Ca 2+ ] i ) dynamic changes of Thapsigargin-inhibited oocyte during activation. b Mitochondrial membrane potential of 1 μM Thapsigargin-inhibited oocytes during activation. c Mitochondrial membrane potential fluorescence intensity of 1 μM Thapsigargin-inhibited oocytes. The green and red curves represent labeling with JC-1, indicating relative fluorescence intensities of low membrane potential and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM Thapsigargin inhibition of oocyte spindle formation e Living cell observation of 1 μM Thapsigargin inhibition of oocyte subcortical F-actin distribution

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes

    doi: 10.1186/s12958-020-00643-7

    Figure Lengend Snippet: Effect of SERCAs inhibitor Thapsigargin on oocyte activation. a Cytoplasmic ([Ca 2+ ] i ) dynamic changes of Thapsigargin-inhibited oocyte during activation. b Mitochondrial membrane potential of 1 μM Thapsigargin-inhibited oocytes during activation. c Mitochondrial membrane potential fluorescence intensity of 1 μM Thapsigargin-inhibited oocytes. The green and red curves represent labeling with JC-1, indicating relative fluorescence intensities of low membrane potential and high membrane potential, respectively. The black curve shows the ratio of high membrane potential to low membrane potential indicating relative mitochondrial membrane potential. d Living cell observation of 1 μM Thapsigargin inhibition of oocyte spindle formation e Living cell observation of 1 μM Thapsigargin inhibition of oocyte subcortical F-actin distribution

    Article Snippet: The details of Ca2+ channel blockers used in the study are described below: Thapsigargin (10,522, Cayman, Michigan, USA), a SERCAs inhibitor [ ] stored in DMSO in 10 mM, with gradient working concentrations of 0.5, 1, and 10 μM; NS-8593 (N-195, Alomone, Jerusalem BioPark, Jerusalem, Israel), a TRPM7 inhibitor [ ], stored in DMSO in 10 mM, with working gradient concentrations of 0.1, 1, and 5 μM; Mibefradil (Mib, HY-15553, MCE, NJ 08852, USA), a T-type Ca2+ channels blocker [ ], stored in water in 10 mM, with working gradient concentrations of 0.5, 5, and 10 μM; GSK-7975A (HY12507, MCE, NJ 08852, USA), an Orai1blocker [ ], stored in DMSO in 10 mM, with gradient concentrations of 10, 100 μM, and 1 mM.

    Techniques: Activation Assay, Fluorescence, Labeling, Inhibition

    IFNα exerts direct cytotoxicity to HBsAg accumulating hepatocytes and downregulate UPR. (a-c) Effects of IFN signaling and UPR modulation on HBsAg accumulating hepatocytes in vitro. (A) Experimental design. (B) LDH levels in the supernatant of PMH culture were measured at indicated time points after adding medium (white bars) or IFNα (0.1MU/ml) (black bars). (C) The immunoblots of UPR related molecules and ISG15 at specified time points after IFNα treatment. (D, E) IFNα suppresses chemically induced UPR in vitro. (D) Experimental schema. (E) Representative immunoblots show the effect of IFNα on UPR related-protein levels at 0, 6, and 12 hours after treatment with thapsigargin.

    Journal: bioRxiv

    Article Title: Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

    doi: 10.1101/2020.12.22.423938

    Figure Lengend Snippet: IFNα exerts direct cytotoxicity to HBsAg accumulating hepatocytes and downregulate UPR. (a-c) Effects of IFN signaling and UPR modulation on HBsAg accumulating hepatocytes in vitro. (A) Experimental design. (B) LDH levels in the supernatant of PMH culture were measured at indicated time points after adding medium (white bars) or IFNα (0.1MU/ml) (black bars). (C) The immunoblots of UPR related molecules and ISG15 at specified time points after IFNα treatment. (D, E) IFNα suppresses chemically induced UPR in vitro. (D) Experimental schema. (E) Representative immunoblots show the effect of IFNα on UPR related-protein levels at 0, 6, and 12 hours after treatment with thapsigargin.

    Article Snippet: Thapsigargin (5µM, Fujifilm-Wako, Osaka, Japan) was added to IFNα-treated WT-PMHs that were then harvested after 6 and 12 hours.

    Techniques: In Vitro, Western Blot

    Peroxisome deficiency does not enhance ER-to-Golgi trafficking of GFP-ATF6(S1P-). Confocal microscopy images show GFP-ATF6(S1P-) trafficking related to the Golgi marker giantin in CHO-K1 and ZR-82 cells. Cells were either cultured in 10% FCS (Control) (A) or treated with 100 nM thapsigargin (B) or 0.5 μg/ml tunicamycin (C) for 12 h. GFP-ATF6 was visualized using a chicken polyclonal anti-GFP antibody; the Golgi was visualized by rabbit anti-giantin; DAPI was used for nuclear staining. Representative fields are shown for each condition. The scale bars represent 10 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Functional Peroxisomes Are Essential for Efficient Cholesterol Sensing and Synthesis

    doi: 10.3389/fcell.2020.560266

    Figure Lengend Snippet: Peroxisome deficiency does not enhance ER-to-Golgi trafficking of GFP-ATF6(S1P-). Confocal microscopy images show GFP-ATF6(S1P-) trafficking related to the Golgi marker giantin in CHO-K1 and ZR-82 cells. Cells were either cultured in 10% FCS (Control) (A) or treated with 100 nM thapsigargin (B) or 0.5 μg/ml tunicamycin (C) for 12 h. GFP-ATF6 was visualized using a chicken polyclonal anti-GFP antibody; the Golgi was visualized by rabbit anti-giantin; DAPI was used for nuclear staining. Representative fields are shown for each condition. The scale bars represent 10 μm.

    Article Snippet: Thapsigargin (BML-PE180; Enzo Life Sciences) was dissolved in DMSO as a 1 mM stock solution and stored at −20°C.

    Techniques: Confocal Microscopy, Marker, Cell Culture, Staining

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

    Journal: The Journal of General Physiology

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

    doi: 10.1085/jgp.200308982

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

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

    Techniques: Fluorescence, Transferring, Injection, Concentration Assay

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

    Journal: The Journal of General Physiology

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

    doi: 10.1085/jgp.200308982

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

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

    Techniques: Activation Assay, Transferring

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

    Journal: Journal of Experimental & Clinical Cancer Research : CR

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

    doi: 10.1186/s13046-018-0935-8

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

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

    Techniques: Concentration Assay, Western Blot

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

    Journal: Arthritis Research & Therapy

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

    doi: 10.1186/s13075-017-1386-x

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

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

    Techniques: Expressing, Real-time Polymerase Chain Reaction

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

    Journal: Arthritis Research & Therapy

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

    doi: 10.1186/s13075-017-1386-x

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

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

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

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

    Journal: Arthritis Research & Therapy

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

    doi: 10.1186/s13075-017-1386-x

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

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

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

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

    Journal: Diabetologia

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

    doi: 10.1007/s00125-011-2181-x

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

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

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

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

    Journal: Diabetologia

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

    doi: 10.1007/s00125-011-2181-x

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

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

    Techniques: Expressing

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

    Journal: Cell Calcium

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

    doi: 10.1016/j.ceca.2015.05.005

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

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

    Techniques: Isolation, Mouse Assay, Incubation

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

    Journal: Cell Calcium

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

    doi: 10.1016/j.ceca.2015.05.005

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

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

    Techniques: Isolation, Mouse Assay, Incubation

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

    Journal: Nature medicine

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

    doi: 10.1038/nm.4166

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

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

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

    Supplemental expression of PIMT reduces EMT and cell invasion in A549 cells induced by Thapsigargin ( A ) Immunoblotting of PIMT and E-cadherin in A549 cells treated with 0.1 µM of Tg, and PIMT expression and empty vectors. ( B, C ) Intensity of PIMT and E-cadherin in A549 cells treated with Tg, and PIMT expression and empty vectors. * indicates p

    Journal: Oncotarget

    Article Title: Deficiency of protein-L-isoaspartate (D-aspartate) O-methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma

    doi: 10.18632/oncotarget.24324

    Figure Lengend Snippet: Supplemental expression of PIMT reduces EMT and cell invasion in A549 cells induced by Thapsigargin ( A ) Immunoblotting of PIMT and E-cadherin in A549 cells treated with 0.1 µM of Tg, and PIMT expression and empty vectors. ( B, C ) Intensity of PIMT and E-cadherin in A549 cells treated with Tg, and PIMT expression and empty vectors. * indicates p

    Article Snippet: Thapsigargin (Tg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques: Expressing

    Increased expression of HIFα and/or Twist in A549 and H441 cells induced by the inhibition of PIMT and Thapsigargin ( A ) Immunoblotting of Slug, ZEB1, Snail1, Twist, and HIF1α in A549 sh-PIMT and sh-control cells. ( B, C ) Immunoblotting and relative intensity of HIF1α in A549 cells treated with Tg. ( D ) Immunoblotting of Slug, ZEB1, Snail1, Twist, and HIF1α in si-control cells and si-PIMT H441 cells. ( E, F ) Immunoblotting and relative intensity of HIF1α in H441 cells treated with Tg. #1 and #2 indicates si-RNA of J-010000-05-0002 and J-010000-07-0002, respectively.

    Journal: Oncotarget

    Article Title: Deficiency of protein-L-isoaspartate (D-aspartate) O-methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma

    doi: 10.18632/oncotarget.24324

    Figure Lengend Snippet: Increased expression of HIFα and/or Twist in A549 and H441 cells induced by the inhibition of PIMT and Thapsigargin ( A ) Immunoblotting of Slug, ZEB1, Snail1, Twist, and HIF1α in A549 sh-PIMT and sh-control cells. ( B, C ) Immunoblotting and relative intensity of HIF1α in A549 cells treated with Tg. ( D ) Immunoblotting of Slug, ZEB1, Snail1, Twist, and HIF1α in si-control cells and si-PIMT H441 cells. ( E, F ) Immunoblotting and relative intensity of HIF1α in H441 cells treated with Tg. #1 and #2 indicates si-RNA of J-010000-05-0002 and J-010000-07-0002, respectively.

    Article Snippet: Thapsigargin (Tg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques: Expressing, Inhibition

    Response of lung adenocarcinoma cell lines to Thapsigargin ( A ) Immunoblotting of GRP78, PIMT, p53, vimentin, and E-cadherin in A549 cells treated with Thapsigargin (Tg). Line 1, DMSO; Line 2, 1.0 × 10 –4 µM of Tg; Line 3, 1.0 × 10 –3 µM of Tg; Line 4, 5.0 × 10 –3 µM of Tg; Line 5, 1.0 × 10 –2 µM of Tg; Line 6, 5.0 × 10 –2 µM of Tg; Line 7, 0.1 µM of Tg; Line 8, 0.2 µM of Tg; Line 9, 0.5 µM of Tg. ( B ) Intensity of GRP78 and p53 in A549 cells treated with Tg. ( C ) Intensity of PIMT and E-cadherin in A549 cells treated with Tg. Immunoblotting and relative intensity of GRP78, PIMT, vimentin, and E-cadherin in H441 ( D–F ), H1650 ( G–I ), and H1650 ( J–L ) cells treated with Tg. Line 1, DMSO; Line 2, 1.0 × 10 –4 µM of Tg; Line 3, 1.0 × 10 –3 µM of Tg; Line 4, 1.0 × 10 –2 µM of Tg; Line 5, 0.1 µM of Tg; Line 6, 1.0 µM of Tg.

    Journal: Oncotarget

    Article Title: Deficiency of protein-L-isoaspartate (D-aspartate) O-methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma

    doi: 10.18632/oncotarget.24324

    Figure Lengend Snippet: Response of lung adenocarcinoma cell lines to Thapsigargin ( A ) Immunoblotting of GRP78, PIMT, p53, vimentin, and E-cadherin in A549 cells treated with Thapsigargin (Tg). Line 1, DMSO; Line 2, 1.0 × 10 –4 µM of Tg; Line 3, 1.0 × 10 –3 µM of Tg; Line 4, 5.0 × 10 –3 µM of Tg; Line 5, 1.0 × 10 –2 µM of Tg; Line 6, 5.0 × 10 –2 µM of Tg; Line 7, 0.1 µM of Tg; Line 8, 0.2 µM of Tg; Line 9, 0.5 µM of Tg. ( B ) Intensity of GRP78 and p53 in A549 cells treated with Tg. ( C ) Intensity of PIMT and E-cadherin in A549 cells treated with Tg. Immunoblotting and relative intensity of GRP78, PIMT, vimentin, and E-cadherin in H441 ( D–F ), H1650 ( G–I ), and H1650 ( J–L ) cells treated with Tg. Line 1, DMSO; Line 2, 1.0 × 10 –4 µM of Tg; Line 3, 1.0 × 10 –3 µM of Tg; Line 4, 1.0 × 10 –2 µM of Tg; Line 5, 0.1 µM of Tg; Line 6, 1.0 µM of Tg.

    Article Snippet: Thapsigargin (Tg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

    Techniques:

    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

    ER stress was activated by TG and DTT in HUVECs. Relative mRNA expression by qPCR of ER stress markers: BiP (A) , CHOP (B) , and TRIB3 (C) in HUVECs treated with thapsigargin (TG; 300 nM, 24 h) and BiP (D) , CHOP (E) , ATF-4 (F) and TRIB3 (G) in HUVECs treated with Dithiothreitol (DTT; 2M, 24 h) normalized against housekeeping gene β -actin ( n = 3–4). (H) , Western blot of protein expression of BiP in HUVECs treated with thapsigargin (TG; 300 nM, 24 h) in the presence or absence of PBA (10 mM). Bars represent pooled densitometry data normalized to total amount of β-actin loading control expressed as fold change compared to control (CTL) ( n = 4 per group). Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA followed with Tukey's multiple comparison test. ** P

    Journal: Frontiers in Cardiovascular Medicine

    Article Title: Endoplasmic Reticulum (ER) Stress-Generated Extracellular Vesicles (Microparticles) Self-Perpetuate ER Stress and Mediate Endothelial Cell Dysfunction Independently of Cell Survival

    doi: 10.3389/fcvm.2020.584791

    Figure Lengend Snippet: ER stress was activated by TG and DTT in HUVECs. Relative mRNA expression by qPCR of ER stress markers: BiP (A) , CHOP (B) , and TRIB3 (C) in HUVECs treated with thapsigargin (TG; 300 nM, 24 h) and BiP (D) , CHOP (E) , ATF-4 (F) and TRIB3 (G) in HUVECs treated with Dithiothreitol (DTT; 2M, 24 h) normalized against housekeeping gene β -actin ( n = 3–4). (H) , Western blot of protein expression of BiP in HUVECs treated with thapsigargin (TG; 300 nM, 24 h) in the presence or absence of PBA (10 mM). Bars represent pooled densitometry data normalized to total amount of β-actin loading control expressed as fold change compared to control (CTL) ( n = 4 per group). Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA followed with Tukey's multiple comparison test. ** P

    Article Snippet: Cell Treatments To pharmacologically induce ER stress, cells were either incubated with thapsigargin (TG) (300 nM, 24 h) (ThermoScientific, Waltham, USA), or Dithiothreitol (DTT) (Sigma-Aldrich, Hamburg, Germany) (2 mM, 24 h).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot