thapsigargin  (Thermo Fisher)


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    Name:
    Thapsigargin
    Description:
    Thapsigargin is a naturally occurring sesquiterpene lactone isolated from the umbelliferous plant Thapsia garganica This tumor promoter releases Ca2 from intracellular stores by specifically inhibiting the endoplasmic reticulum Ca2 ATPase it does not directly affect plasma membrane Ca2 ATPases Ins 1 4 5 P3 production or protein kinase C activity Thapsigargin is also available as a special packaging T7459
    Catalog Number:
    t7458
    Price:
    None
    Applications:
    Cell Analysis|Cellular Imaging|Immunofluorescence (IF)|Immunofluorescence Staining & Detection|Receptors|Cell Structure
    Category:
    Labeling Detection Products
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    Structured Review

    Thermo Fisher thapsigargin
    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or <t>thapsigargin</t> (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P
    Thapsigargin is a naturally occurring sesquiterpene lactone isolated from the umbelliferous plant Thapsia garganica This tumor promoter releases Ca2 from intracellular stores by specifically inhibiting the endoplasmic reticulum Ca2 ATPase it does not directly affect plasma membrane Ca2 ATPases Ins 1 4 5 P3 production or protein kinase C activity Thapsigargin is also available as a special packaging T7459
    https://www.bioz.com/result/thapsigargin/product/Thermo Fisher
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family"

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    Journal: Nature cell biology

    doi: 10.1038/ncb3236

    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P
    Figure Legend Snippet: BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Techniques Used: Transformation Assay, Infection, Expressing, shRNA, Luciferase, Staining, Transfection

    Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P
    Figure Legend Snippet: Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Techniques Used: Quantitative RT-PCR, Generated, Cell Culture, Staining, Mouse Assay, Irradiation, TUNEL Assay, Light Microscopy, Purification

    Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P
    Figure Legend Snippet: Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Techniques Used: Transformation Assay, Cell Culture, Irradiation, Staining

    2) Product Images from "Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells"

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

    Journal: Oncotarget

    doi:

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

    Techniques Used: Produced, Planar Chromatography

    3) Product Images from "Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response"

    Article Title: Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.3.1365-1377.2004

    ATF3 facilitates enhanced GADD34 expression in response to ER stress and amino acid starvation. ATF3 +/+ and ATF3 −/− MEF cells were treated with thapsigargin (Tg) (A), subjected to leucine starvation (B) for the indicated number of hours, or left untreated (0 h). The same amount of protein lysate was analyzed in each lane, and levels of ATF3, ATF4, CHOP, total eIF2α, phosphorylated eIF2α (eIF2α-P), GADD34, and actin were measured as indicated by immunoblot analysis.
    Figure Legend Snippet: ATF3 facilitates enhanced GADD34 expression in response to ER stress and amino acid starvation. ATF3 +/+ and ATF3 −/− MEF cells were treated with thapsigargin (Tg) (A), subjected to leucine starvation (B) for the indicated number of hours, or left untreated (0 h). The same amount of protein lysate was analyzed in each lane, and levels of ATF3, ATF4, CHOP, total eIF2α, phosphorylated eIF2α (eIF2α-P), GADD34, and actin were measured as indicated by immunoblot analysis.

    Techniques Used: Expressing

    Increased expression of ATF3 in response to ER or nutritional stress requires ATF4. ATF4 +/+ and ATF4 −/− MEF cells were treated with thapsigargin (Tg) (A), subjected to leucine starvation (B) for the indicated number of hours, or left untreated (0 h). The same amount of protein lysate was analyzed in each lane, and levels of ATF3, ATF4, CHOP, and actin were measured as indicated by immunoblot analysis with protein-specific antibodies.
    Figure Legend Snippet: Increased expression of ATF3 in response to ER or nutritional stress requires ATF4. ATF4 +/+ and ATF4 −/− MEF cells were treated with thapsigargin (Tg) (A), subjected to leucine starvation (B) for the indicated number of hours, or left untreated (0 h). The same amount of protein lysate was analyzed in each lane, and levels of ATF3, ATF4, CHOP, and actin were measured as indicated by immunoblot analysis with protein-specific antibodies.

    Techniques Used: Expressing

    Overexpression of ATF3 reduces the levels of eIF2α phosphorylation during ER stress. ATF3 was transiently overexpressed in PEK +/+ MEF and HEK 293T cells, and these transfected cells or control PEK −/− cells were treated with thapsigargin (Tg) (+) for 6 h or not subjected to ER stress (−). ATF3 + indicates that this transcription factor was expressed in the indicated cell line, and ATF3 − indicates that only the expression vector was introduced into the cells. Levels of phosphorylated eIF2α (eIF2α-P), total eIF2α, ATF3, GADD34, and actin were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. In PEK +/+ MEF cells, the level of endogenous ATF3 induced by ER stress was lower than that measured in HEK 293T cells.
    Figure Legend Snippet: Overexpression of ATF3 reduces the levels of eIF2α phosphorylation during ER stress. ATF3 was transiently overexpressed in PEK +/+ MEF and HEK 293T cells, and these transfected cells or control PEK −/− cells were treated with thapsigargin (Tg) (+) for 6 h or not subjected to ER stress (−). ATF3 + indicates that this transcription factor was expressed in the indicated cell line, and ATF3 − indicates that only the expression vector was introduced into the cells. Levels of phosphorylated eIF2α (eIF2α-P), total eIF2α, ATF3, GADD34, and actin were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. In PEK +/+ MEF cells, the level of endogenous ATF3 induced by ER stress was lower than that measured in HEK 293T cells.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation

    Enhanced DNA binding by ATF4-C/EBP during ER stress requires PEK. (A to D) Nuclear lysates were prepared from PEK +/+ and PEK −/− MEF cells treated with thapsigargin (Tg) for the indicated time or in the absence of this ER stress agent (0 h). The same amount of lysate was used in EMSAs with radiolabeled double-stranded oligonucleotides containing an ATF-C/EBP composite binding site (A), an OCT1 binding site (B and D), or a GADD34 ATF-CREB binding site (C and E). Binding mixtures were separated by electrophoresis, and bound DNAs were visualized by autoradiographic exposures of the dried polyacrylamide gels. Bound DNA fragments in the EMSAs are indicated by an arrow. Competition indicates that nonradiolabeled competitor DNA was added at a 10× or 100× molar excess. (A) Supershift indicates that an antibody that specifically recognizes ATF3, ATF4, or CHOP was added to the EMSA binding mixture containing the nuclear lysate from PEK +/+ cells subjected to 6 h of ER stress. (C) In lanes 13 and 14, antibody specific to ATF3 or ATF4 was added to the EMSA binding mixture containing nuclear lysates prepared from PEK +/+ MEF cells subjected to 360 min of ER stress. (E) Nuclear lysates were prepared from ATF3 +/+ and ATF3 −/− MEF cells subjected to ER stress (Tg) or to leucine deprivation (−Leu) for the indicated number of hours and assessed for binding to the GADD34 ATF-CREB binding site by EMSA. FP (free probe) indicates the GADD34 ATF-CREB DNA fragment without nuclear lysate. Competition (Comp) indicates that nonradiolabeled ATF-CREB competitor DNA was added at a 10× or 100× molar excess. Supershift experiments with ATF4, ATF3, or CHOP antibodies suggest that only ATF4 is present in the protein-DNA complexes with nuclear lysates prepared from leucine-deprived MEF cells (data not shown).
    Figure Legend Snippet: Enhanced DNA binding by ATF4-C/EBP during ER stress requires PEK. (A to D) Nuclear lysates were prepared from PEK +/+ and PEK −/− MEF cells treated with thapsigargin (Tg) for the indicated time or in the absence of this ER stress agent (0 h). The same amount of lysate was used in EMSAs with radiolabeled double-stranded oligonucleotides containing an ATF-C/EBP composite binding site (A), an OCT1 binding site (B and D), or a GADD34 ATF-CREB binding site (C and E). Binding mixtures were separated by electrophoresis, and bound DNAs were visualized by autoradiographic exposures of the dried polyacrylamide gels. Bound DNA fragments in the EMSAs are indicated by an arrow. Competition indicates that nonradiolabeled competitor DNA was added at a 10× or 100× molar excess. (A) Supershift indicates that an antibody that specifically recognizes ATF3, ATF4, or CHOP was added to the EMSA binding mixture containing the nuclear lysate from PEK +/+ cells subjected to 6 h of ER stress. (C) In lanes 13 and 14, antibody specific to ATF3 or ATF4 was added to the EMSA binding mixture containing nuclear lysates prepared from PEK +/+ MEF cells subjected to 360 min of ER stress. (E) Nuclear lysates were prepared from ATF3 +/+ and ATF3 −/− MEF cells subjected to ER stress (Tg) or to leucine deprivation (−Leu) for the indicated number of hours and assessed for binding to the GADD34 ATF-CREB binding site by EMSA. FP (free probe) indicates the GADD34 ATF-CREB DNA fragment without nuclear lysate. Competition (Comp) indicates that nonradiolabeled ATF-CREB competitor DNA was added at a 10× or 100× molar excess. Supershift experiments with ATF4, ATF3, or CHOP antibodies suggest that only ATF4 is present in the protein-DNA complexes with nuclear lysates prepared from leucine-deprived MEF cells (data not shown).

    Techniques Used: Binding Assay, Electrophoresis

    Enhanced expression of ATF3, ATF4, and CHOP in response to ER stress requires PEK activity. PEK +/+ and PEK −/− MEF cells were exposed to the presence of thapsigargin (Tg) for the indicated number of hours or to the absence of this ER stress agent (0 h). (A) Whole-cell lysates were prepared from the cultured cells, and the levels of eIF2α specifically phosphorylated at Ser-51 (eIF2α-P) or total eIF2α were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. Relative levels of phosphorylated eIF2α were normalized to levels of total eIF2α in each lysate preparation. (B) MEF cells containing functional eIF2 kinases (wildtype, lanes 1 to 4 and 13; PEK −/− GCN2 −/− , lanes 5 to 8; PEK −/− GCN2 −/− PKR −/− , lanes 9 to 12; PEK −/− , lane 14) were treated with thapsigargin for the indicated number of hours. Levels of phosphorylated eIF2α were measured by immunoblot assay. (C) Levels of ATF3, ATF4, CHOP, GRP78, and actin were measured by immunoblot analysis with an antibody specific to each protein. To facilitate normalization between PEK +/+ and PEK −/− panels, the lysate from PEK −/− cells that were stressed for 6 h was included in lane C of the PEK +/+ panel. Similarly, lane C in the PEK −/− immunoblot panel was an analysis of lysate prepared from PEK +/+ MEF cells treated with thapsigargin for 6 h. (D) A plasmid expressing PEK was transiently transfected into PEK −/− MEF cells (lanes 5 and 6), and PEK +/+ and transfected cells were subjected to the presence (+) or absence (−) of ER stress for 6 h. Vec indicates that the parent expression plasmid alone was introduced into the PEK −/− MEF cells (lanes 3 and 4). Whole-cell lysates were prepared from these transfected cells, and ATF3, ATF4, CHOP, PEK, and actin were measured by immunoblot analysis.
    Figure Legend Snippet: Enhanced expression of ATF3, ATF4, and CHOP in response to ER stress requires PEK activity. PEK +/+ and PEK −/− MEF cells were exposed to the presence of thapsigargin (Tg) for the indicated number of hours or to the absence of this ER stress agent (0 h). (A) Whole-cell lysates were prepared from the cultured cells, and the levels of eIF2α specifically phosphorylated at Ser-51 (eIF2α-P) or total eIF2α were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. Relative levels of phosphorylated eIF2α were normalized to levels of total eIF2α in each lysate preparation. (B) MEF cells containing functional eIF2 kinases (wildtype, lanes 1 to 4 and 13; PEK −/− GCN2 −/− , lanes 5 to 8; PEK −/− GCN2 −/− PKR −/− , lanes 9 to 12; PEK −/− , lane 14) were treated with thapsigargin for the indicated number of hours. Levels of phosphorylated eIF2α were measured by immunoblot assay. (C) Levels of ATF3, ATF4, CHOP, GRP78, and actin were measured by immunoblot analysis with an antibody specific to each protein. To facilitate normalization between PEK +/+ and PEK −/− panels, the lysate from PEK −/− cells that were stressed for 6 h was included in lane C of the PEK +/+ panel. Similarly, lane C in the PEK −/− immunoblot panel was an analysis of lysate prepared from PEK +/+ MEF cells treated with thapsigargin for 6 h. (D) A plasmid expressing PEK was transiently transfected into PEK −/− MEF cells (lanes 5 and 6), and PEK +/+ and transfected cells were subjected to the presence (+) or absence (−) of ER stress for 6 h. Vec indicates that the parent expression plasmid alone was introduced into the PEK −/− MEF cells (lanes 3 and 4). Whole-cell lysates were prepared from these transfected cells, and ATF3, ATF4, CHOP, PEK, and actin were measured by immunoblot analysis.

    Techniques Used: Expressing, Activity Assay, Cell Culture, Functional Assay, Plasmid Preparation, Transfection

    mRNA and protein synthesis are required for induced expression of ATF3, ATF4, and CHOP in response to ER stress. (A) PEK +/+ MEF cells were treated with thapsigargin (Tg) and/or cycloheximide (CHX) for the indicated number of hours or not treated (0 h). (B) MEF cells containing functional eIF2 kinases (wild type, lanes 1 to 4; PEK −/− GCN2 −/− , lanes 5 to 8; PEK −/− GCN2 −/− PKR −/− , lanes 9 to 12) were treated with cycloheximide for the indicated number of hours. Levels of phosphorylated eIF2α were measured by immunoblot assay. (C) PEK +/+ MEF cells were treated with thapsigargin and/or actinomycin D (AD) for 3 or 6 h or not treated (0 h). Cell lysates were prepared from the cultured cells, and immunoblot analysis was used to measure the levels of ATF3, ATF4, CHOP, phosphorylated eIF2α (eIF2α-P), and total eIF2α. The same amount of protein lysate was analyzed in each lane.
    Figure Legend Snippet: mRNA and protein synthesis are required for induced expression of ATF3, ATF4, and CHOP in response to ER stress. (A) PEK +/+ MEF cells were treated with thapsigargin (Tg) and/or cycloheximide (CHX) for the indicated number of hours or not treated (0 h). (B) MEF cells containing functional eIF2 kinases (wild type, lanes 1 to 4; PEK −/− GCN2 −/− , lanes 5 to 8; PEK −/− GCN2 −/− PKR −/− , lanes 9 to 12) were treated with cycloheximide for the indicated number of hours. Levels of phosphorylated eIF2α were measured by immunoblot assay. (C) PEK +/+ MEF cells were treated with thapsigargin and/or actinomycin D (AD) for 3 or 6 h or not treated (0 h). Cell lysates were prepared from the cultured cells, and immunoblot analysis was used to measure the levels of ATF3, ATF4, CHOP, phosphorylated eIF2α (eIF2α-P), and total eIF2α. The same amount of protein lysate was analyzed in each lane.

    Techniques Used: Expressing, Functional Assay, Cell Culture

    ATF3 and CHOP are induced by eIF2 kinase GCN2 in response to amino acid depletion. GCN2 +/+ and GCN2 −/− MEF cells were grown in leucine-depleted medium for the indicated number of hours, exposed to thapsigargin (Tg), or subjected to no stress. Whole-cell lysates were prepared from the cultured cells, and the levels of phosphorylated eIF2α (eIF2α-P), total eIF2α, ATF3, ATF4, and CHOP were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. Relative levels of phosphorylated eIF2α or ATF3 under the indicated stress condition were normalized to levels of total eIF2α in each lysate preparation.
    Figure Legend Snippet: ATF3 and CHOP are induced by eIF2 kinase GCN2 in response to amino acid depletion. GCN2 +/+ and GCN2 −/− MEF cells were grown in leucine-depleted medium for the indicated number of hours, exposed to thapsigargin (Tg), or subjected to no stress. Whole-cell lysates were prepared from the cultured cells, and the levels of phosphorylated eIF2α (eIF2α-P), total eIF2α, ATF3, ATF4, and CHOP were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. Relative levels of phosphorylated eIF2α or ATF3 under the indicated stress condition were normalized to levels of total eIF2α in each lysate preparation.

    Techniques Used: Cell Culture

    ATF3 facilitates expression of GADD34 in response to ER stress. ATF3 was transiently expressed in PEK −/− MEF cells, and PEK +/+ and transfected (tsf) cells were subjected to the presence (+) or absence (−) of ER stress for the indicated number of hours. Vec indicates that the parent expression plasmid alone was introduced into the PEK −/− MEF cells. Whole-cell lysates were prepared from these transfected cells, and phosphorylated eIF2α (eIF2α-P), total eIF2α, and GADD34 levels were measured by immunoblot analysis. Tg, thapsigargin.
    Figure Legend Snippet: ATF3 facilitates expression of GADD34 in response to ER stress. ATF3 was transiently expressed in PEK −/− MEF cells, and PEK +/+ and transfected (tsf) cells were subjected to the presence (+) or absence (−) of ER stress for the indicated number of hours. Vec indicates that the parent expression plasmid alone was introduced into the PEK −/− MEF cells. Whole-cell lysates were prepared from these transfected cells, and phosphorylated eIF2α (eIF2α-P), total eIF2α, and GADD34 levels were measured by immunoblot analysis. Tg, thapsigargin.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    4) Product Images from "Up-regulation of Orai1 expression and store operated Ca2+ entry following activation of membrane androgen receptors in MCF-7 breast tumor cells"

    Article Title: Up-regulation of Orai1 expression and store operated Ca2+ entry following activation of membrane androgen receptors in MCF-7 breast tumor cells

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-2014-2

    Effect of mAR activation on intracellular Ca 2+ release and store operated Ca 2+ entry (SOCE) in MCF-7 cells. a Representative tracings of fura-2 fluorescence-ratio in fluorescence spectrometry before, during and after Ca 2+ depletion with subsequent addition of thapsigargin (1 μM) in MCF-7 cells without (control, open squares) and with (grey and black squares) treatment with testosterone-albumin-conjugates (TAC, 100 nM) for 15–120 min in the absence and presence of the Orai-1 inhibitor 2-APB (50 μM). b , c Arithmetic means (± SEM, n = 3–5, each experiment 10–30 cells) of slope ( b ) and peak ( c ) increase of fura-2-fluorescence-ratio following re-addition of extracellular Ca 2+ in MCF-7 cells without (control, white bars) and with (grey and black bars) treatment with TAC (100 nM) for 15–120 min in the absence and presence of the Orai-1 inhibitor 2-APB (50 μM). ***( p
    Figure Legend Snippet: Effect of mAR activation on intracellular Ca 2+ release and store operated Ca 2+ entry (SOCE) in MCF-7 cells. a Representative tracings of fura-2 fluorescence-ratio in fluorescence spectrometry before, during and after Ca 2+ depletion with subsequent addition of thapsigargin (1 μM) in MCF-7 cells without (control, open squares) and with (grey and black squares) treatment with testosterone-albumin-conjugates (TAC, 100 nM) for 15–120 min in the absence and presence of the Orai-1 inhibitor 2-APB (50 μM). b , c Arithmetic means (± SEM, n = 3–5, each experiment 10–30 cells) of slope ( b ) and peak ( c ) increase of fura-2-fluorescence-ratio following re-addition of extracellular Ca 2+ in MCF-7 cells without (control, white bars) and with (grey and black bars) treatment with TAC (100 nM) for 15–120 min in the absence and presence of the Orai-1 inhibitor 2-APB (50 μM). ***( p

    Techniques Used: Activation Assay, Fluorescence

    5) Product Images from "Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells "

    Article Title: Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

    Journal: The Journal of General Physiology

    doi:

    Effect of Ca 2+ -store depletion on GR-GFP import HM1 cells. (A) Quantification of Dex-induced GR-GFP translocation in transfected HM1 cells. The ratio of nuclear/cytoplasmic fluorescence was determined without treatment (left) or after a 30-min pretreatment with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), 1 μM ionomycin (Iono), or 10 μM BAPTA-AM and subsequent incubation with 1 μM Dex for 20 min (right). The nuclear/cytoplasmic fluorescence ratio after Dex application was significantly reduced by BAPTA-AM pretreatment (*** P ≤ 0.001; analysis of variance). All experiments were performed in Ca 2+ -free buffer. Bars represent means ± SEM. n, number of experiments; m, number of cells in each experiment. (B) Representative fluorescence images of HM1 cells 20 min after application of 1 μM Dex. Cells were pretreated with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), or 10 μM BAPTA-AM as in A.
    Figure Legend Snippet: Effect of Ca 2+ -store depletion on GR-GFP import HM1 cells. (A) Quantification of Dex-induced GR-GFP translocation in transfected HM1 cells. The ratio of nuclear/cytoplasmic fluorescence was determined without treatment (left) or after a 30-min pretreatment with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), 1 μM ionomycin (Iono), or 10 μM BAPTA-AM and subsequent incubation with 1 μM Dex for 20 min (right). The nuclear/cytoplasmic fluorescence ratio after Dex application was significantly reduced by BAPTA-AM pretreatment (*** P ≤ 0.001; analysis of variance). All experiments were performed in Ca 2+ -free buffer. Bars represent means ± SEM. n, number of experiments; m, number of cells in each experiment. (B) Representative fluorescence images of HM1 cells 20 min after application of 1 μM Dex. Cells were pretreated with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), or 10 μM BAPTA-AM as in A.

    Techniques Used: Translocation Assay, Transfection, Fluorescence, Incubation

    Independence of GFP-MK2 export of Ca 2+ -store depletion and calmodulin. (A) Effect of store-depleting agents and calmidazolium on Aniso-induced export of GFP-MK2. Cells were pretreated with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), 1 μM ionomycin (Iono), ionomycin plus 5 μM calmidazolium (Iono + Calmid), or 10 μM BAPTA-AM for 30 min and subsequently incubated with 10 μg/ml Aniso. The Aniso-induced change in NF/CF in control cells was considered as 100% export activity for each experiment. Data represent weighted means ± SEM from three experiments. m, number of cells in each experiment. No pretreatment was found to significantly decrease GFP-MK2 export. In two of three experiments, export was significantly stimulated ( P ≤ 0.05, t test) by carbachol pretreatment. All experiments were carried out in Ca 2+ -free buffer. (B) Representative fluorescence images of cells 20 min after application of 10 μg/ml Aniso. Cells were pretreated with thapsigargin (Tg), carbachol (Carb), or BAPTA-AM, as in A.
    Figure Legend Snippet: Independence of GFP-MK2 export of Ca 2+ -store depletion and calmodulin. (A) Effect of store-depleting agents and calmidazolium on Aniso-induced export of GFP-MK2. Cells were pretreated with 1 μM thapsigargin (Tg), 10 μM carbachol (Carb), 1 μM ionomycin (Iono), ionomycin plus 5 μM calmidazolium (Iono + Calmid), or 10 μM BAPTA-AM for 30 min and subsequently incubated with 10 μg/ml Aniso. The Aniso-induced change in NF/CF in control cells was considered as 100% export activity for each experiment. Data represent weighted means ± SEM from three experiments. m, number of cells in each experiment. No pretreatment was found to significantly decrease GFP-MK2 export. In two of three experiments, export was significantly stimulated ( P ≤ 0.05, t test) by carbachol pretreatment. All experiments were carried out in Ca 2+ -free buffer. (B) Representative fluorescence images of cells 20 min after application of 10 μg/ml Aniso. Cells were pretreated with thapsigargin (Tg), carbachol (Carb), or BAPTA-AM, as in A.

    Techniques Used: Incubation, Activity Assay, Fluorescence

    Nuclear localization of a nuclear-targeted fluorescent marker (pcDIC15nu) does not differ between control and Ca 2+ -depleted HM1 cells. Fluorescence (left) and overlay images (right) of pcDIC15nu-expressing cells kept in normal growth medium (top) or Ca 2+ -free medium supplemented with 100 nM thapsigargin and 5 μM calmidazolium (bottom) for 12 h after transfection.
    Figure Legend Snippet: Nuclear localization of a nuclear-targeted fluorescent marker (pcDIC15nu) does not differ between control and Ca 2+ -depleted HM1 cells. Fluorescence (left) and overlay images (right) of pcDIC15nu-expressing cells kept in normal growth medium (top) or Ca 2+ -free medium supplemented with 100 nM thapsigargin and 5 μM calmidazolium (bottom) for 12 h after transfection.

    Techniques Used: Marker, Fluorescence, Expressing, Transfection

    6) Product Images from "Mechanisms regulating cytochrome c release in pancreatic mitochondria"

    Article Title: Mechanisms regulating cytochrome c release in pancreatic mitochondria

    Journal: Gut

    doi: 10.1136/gut.2007.147207

    Cholecystokinin (CCK)-induced apoptosis in pancreatic acinar cells is mediated through both Ca 2+ -dependent and -independent mechanisms. (A) Isolated rat pancreatic acinar cells were incubated for 3 h with and without CCK-8 (100 nmol/l) or thapsigargin
    Figure Legend Snippet: Cholecystokinin (CCK)-induced apoptosis in pancreatic acinar cells is mediated through both Ca 2+ -dependent and -independent mechanisms. (A) Isolated rat pancreatic acinar cells were incubated for 3 h with and without CCK-8 (100 nmol/l) or thapsigargin

    Techniques Used: Isolation, Incubation, CCK-8 Assay

    7) Product Images from "Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons"

    Article Title: Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.297

    N-Bak protein is not induced in the apoptotic or stress conditions. ( a ) The 5 DIV SCG neurons were deprived of NGF for 2 days in the presence of caspase inhibitor BAF, or not deprived. The 6 DIV cortical neurons were treated for 24 h with 4 μ g/ml of etoposide or for 1 h with 10 nM of thapsigargin in the presence of caspase inhibitor Q-VD-OPh, or left untreated. Immunoblot was probed with anti-Bak antibodies. The position of ∼20 kD N-Bak is shown on the lane of HeLa cells overexpressing the apoptotically inactive L76E mutant. Note the absence of ∼20 kD N-Bak protein in the SCG neurons and the presence of ∼21 kD nonspecific band on the lanes of cortical neurons. The boxed areas were cut from the parallel gel and analyzed by mass spectrometry. ( b ) Cortical neurons were treated with 10 nM of thapsigargin in the presence of caspase inhibitor QVD-OPh for the indicated times, or not treated (NT). Immunoblot was probed with antibodies to PeIF2 α (upper panel), known to be increasingly phosphorylated in the endoplasmic reticulum stress, and reprobed with anti-Bak antibodies (lower panel). The position of ∼20 kD N-Bak is shown on the lane of HeLa cells overexpressing the apoptotically inactive L76E mutant (HeLa-N-BakL76E). The Bak protein of ∼25 kD in ( a ) and ( b ) comes from the nonneuronal cells in the cultures and its signal is overexposed
    Figure Legend Snippet: N-Bak protein is not induced in the apoptotic or stress conditions. ( a ) The 5 DIV SCG neurons were deprived of NGF for 2 days in the presence of caspase inhibitor BAF, or not deprived. The 6 DIV cortical neurons were treated for 24 h with 4 μ g/ml of etoposide or for 1 h with 10 nM of thapsigargin in the presence of caspase inhibitor Q-VD-OPh, or left untreated. Immunoblot was probed with anti-Bak antibodies. The position of ∼20 kD N-Bak is shown on the lane of HeLa cells overexpressing the apoptotically inactive L76E mutant. Note the absence of ∼20 kD N-Bak protein in the SCG neurons and the presence of ∼21 kD nonspecific band on the lanes of cortical neurons. The boxed areas were cut from the parallel gel and analyzed by mass spectrometry. ( b ) Cortical neurons were treated with 10 nM of thapsigargin in the presence of caspase inhibitor QVD-OPh for the indicated times, or not treated (NT). Immunoblot was probed with antibodies to PeIF2 α (upper panel), known to be increasingly phosphorylated in the endoplasmic reticulum stress, and reprobed with anti-Bak antibodies (lower panel). The position of ∼20 kD N-Bak is shown on the lane of HeLa cells overexpressing the apoptotically inactive L76E mutant (HeLa-N-BakL76E). The Bak protein of ∼25 kD in ( a ) and ( b ) comes from the nonneuronal cells in the cultures and its signal is overexposed

    Techniques Used: Mutagenesis, Mass Spectrometry

    8) Product Images from "Regulation of Calreticulin Gene Expression by Calcium "

    Article Title: Regulation of Calreticulin Gene Expression by Calcium

    Journal: The Journal of Cell Biology

    doi:

    Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with DMSO ( Control ), 10 μM A23187 ( A23187 ), or 100 nM thapsigargin ( Thapsigargin ), and transcription was allowed to proceed in the presence of [ 32 P]UTP as described in Materials and Methods. RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect sense ( S ) or antisense ( AS ) transcript arising from calreticulin, actin, G3PDH, H2b, and c- myc genes. CRT-1 , calreticulin 5″ probe; CRT-2 , calreticulin 3″ probe; actin-1 , γ-actin 3″ probe; actin-2 , γ-actin 5″ probe.
    Figure Legend Snippet: Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with DMSO ( Control ), 10 μM A23187 ( A23187 ), or 100 nM thapsigargin ( Thapsigargin ), and transcription was allowed to proceed in the presence of [ 32 P]UTP as described in Materials and Methods. RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect sense ( S ) or antisense ( AS ) transcript arising from calreticulin, actin, G3PDH, H2b, and c- myc genes. CRT-1 , calreticulin 5″ probe; CRT-2 , calreticulin 3″ probe; actin-1 , γ-actin 3″ probe; actin-2 , γ-actin 5″ probe.

    Techniques Used: Isolation

    Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were loaded with 10 μM BAPTA/AM for 30 min in a media containing different drugs for 16 h and lysed, and reporter gene analysis was carried out as described in Materials and Methods. ( Open bars ) Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; ( dotted bars ) cells loaded with BAPTA/AM followed by incubation with either A23187 or thapsigargin (control cells were not treated with BAPTA/AM); ( filled bars ) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca 2+ -depleted DME. ( Cross-hatched bars ) Cells loaded with 10 μM EGTA/AM for 30 min followed by 16-h incubation with either A23187 or thaps igargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.
    Figure Legend Snippet: Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were loaded with 10 μM BAPTA/AM for 30 min in a media containing different drugs for 16 h and lysed, and reporter gene analysis was carried out as described in Materials and Methods. ( Open bars ) Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; ( dotted bars ) cells loaded with BAPTA/AM followed by incubation with either A23187 or thapsigargin (control cells were not treated with BAPTA/AM); ( filled bars ) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca 2+ -depleted DME. ( Cross-hatched bars ) Cells loaded with 10 μM EGTA/AM for 30 min followed by 16-h incubation with either A23187 or thaps igargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.

    Techniques Used: Activation Assay, Incubation

    Cycloheximide inhibits the A23187- and thapsigargin-dependent increase of calreticulin mRNA levels. Total RNA was isolated from nontransfected NIH/3T3 cells that had been incubated for 16–18 h with 7 μM A23187 or 100 nM thapsigargin, and either with or without cycloheximide. The RNA was separated electrophoretically on a formaldehyde-agarose gel, blotted onto Hybond N nylon filters, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH as described in Materials and Methods. ( Top ) Autoradiogram of a Northern blot probed with calreticulin and the G3PDH cDNAs. (Lane 1 ) Control cells; (lane 2 ) cells incazxubated with 100 μM cycloheximide; (lane 3 ) A23187-treated cells; (lane 4 ) A23187- and cycloheximide-treated cells; (lane 5 ) thapsigargin-treated cells; (lane 6 ) thapsigargin- and cycloheximide-treated cells. ( Bottom ) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of the Northern blots. ( Filled bars ) Cells treated with A23187 or thapsigargin in the absence of cycloheximide; ( open bars ) cells pretreated with 100 μM cycloheximide for 2 h followed by incubation with either A23817 or thapsigargin. TG , thapsigargin.
    Figure Legend Snippet: Cycloheximide inhibits the A23187- and thapsigargin-dependent increase of calreticulin mRNA levels. Total RNA was isolated from nontransfected NIH/3T3 cells that had been incubated for 16–18 h with 7 μM A23187 or 100 nM thapsigargin, and either with or without cycloheximide. The RNA was separated electrophoretically on a formaldehyde-agarose gel, blotted onto Hybond N nylon filters, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH as described in Materials and Methods. ( Top ) Autoradiogram of a Northern blot probed with calreticulin and the G3PDH cDNAs. (Lane 1 ) Control cells; (lane 2 ) cells incazxubated with 100 μM cycloheximide; (lane 3 ) A23187-treated cells; (lane 4 ) A23187- and cycloheximide-treated cells; (lane 5 ) thapsigargin-treated cells; (lane 6 ) thapsigargin- and cycloheximide-treated cells. ( Bottom ) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of the Northern blots. ( Filled bars ) Cells treated with A23187 or thapsigargin in the absence of cycloheximide; ( open bars ) cells pretreated with 100 μM cycloheximide for 2 h followed by incubation with either A23817 or thapsigargin. TG , thapsigargin.

    Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Northern Blot

    Effects of bradykinin on activation of calreticulin promoter. NCB1 cells were incubated with either 100 nM thapsigargin ( TG ) or 200 nM bradykinin ( BK ) in Ca 2+ -depleted DME supplemented with EGTA. After 16 h the cells were lysed, and then reporter gene analysis was carried out as described in Materials and Methods. Data are shown as a mean ± SD.
    Figure Legend Snippet: Effects of bradykinin on activation of calreticulin promoter. NCB1 cells were incubated with either 100 nM thapsigargin ( TG ) or 200 nM bradykinin ( BK ) in Ca 2+ -depleted DME supplemented with EGTA. After 16 h the cells were lysed, and then reporter gene analysis was carried out as described in Materials and Methods. Data are shown as a mean ± SD.

    Techniques Used: Activation Assay, Incubation

    Activation of the calreticulin promoter by A23187 and thapsigargin treatment. ( A ) NIH/3T3 were stably transfected with either pCC1 or pLC1 and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or an appropriate volume of DMSO (control cells), as described in Materials and Methods. ( B ) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the 2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the calreticulin promoter, respectively. NIH/3T3 were transiently transfected with different pCC vectors and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187 ( open bars ) or 300 nM thapsigargin ( filled bars ) and lysed, and reporter enzyme assays were carried out. n / d , not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.
    Figure Legend Snippet: Activation of the calreticulin promoter by A23187 and thapsigargin treatment. ( A ) NIH/3T3 were stably transfected with either pCC1 or pLC1 and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or an appropriate volume of DMSO (control cells), as described in Materials and Methods. ( B ) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the 2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the calreticulin promoter, respectively. NIH/3T3 were transiently transfected with different pCC vectors and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187 ( open bars ) or 300 nM thapsigargin ( filled bars ) and lysed, and reporter enzyme assays were carried out. n / d , not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.

    Techniques Used: Activation Assay, Stable Transfection, Transfection, Incubation, Expressing, Plasmid Preparation, Periodic Counter-current Chromatography

    A23187- and thapsigargin-dependent increase in calreticulin mRNA levels ( A ) and protein levels ( B ). ( A ) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or DMSO. Total RNA was then isolated and electrophoretically separated on a formaldehyde-agarose gel, blotted onto Hybond N nylon membrane, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. ( Top ) Autoradiogram of a Northern blot probed with calreticulin and G3PDH cDNA. (Lane 1 ) Control cells; (lane 2 ) A23187-treated cells; (lane 3 ) thapsigargin-treated cells. ( Bottom ) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. ( B ) NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin and cellular extracts were prepared as described by Mery et al. (1996) . The proteins were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membranes, and incubated with goat anti-calreticulin antibody as described by Milner et al. (1991) . For each condition, three different amounts of cells were analyzed (lanes 1 and 4 , 30,000 cells per well; lanes 2 and 5 , 100,000 cells per well; lanes 3 and 6 , 150,000 cells per well), and the immunoblots were scanned by densitometry. ( Top , lanes 1 , 2 , and 3 ) Control, untreated cells; (lanes 4 , 5 , and 6 ) thapsigargin-treated cells. ( Bottom ) The slope of the cell number as a function of OD was obtained by a linear fit to give a relative cellular content of calreticulin ( Mery et al., 1996 ). TG , thapsigargin.
    Figure Legend Snippet: A23187- and thapsigargin-dependent increase in calreticulin mRNA levels ( A ) and protein levels ( B ). ( A ) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or DMSO. Total RNA was then isolated and electrophoretically separated on a formaldehyde-agarose gel, blotted onto Hybond N nylon membrane, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. ( Top ) Autoradiogram of a Northern blot probed with calreticulin and G3PDH cDNA. (Lane 1 ) Control cells; (lane 2 ) A23187-treated cells; (lane 3 ) thapsigargin-treated cells. ( Bottom ) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. ( B ) NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin and cellular extracts were prepared as described by Mery et al. (1996) . The proteins were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membranes, and incubated with goat anti-calreticulin antibody as described by Milner et al. (1991) . For each condition, three different amounts of cells were analyzed (lanes 1 and 4 , 30,000 cells per well; lanes 2 and 5 , 100,000 cells per well; lanes 3 and 6 , 150,000 cells per well), and the immunoblots were scanned by densitometry. ( Top , lanes 1 , 2 , and 3 ) Control, untreated cells; (lanes 4 , 5 , and 6 ) thapsigargin-treated cells. ( Bottom ) The slope of the cell number as a function of OD was obtained by a linear fit to give a relative cellular content of calreticulin ( Mery et al., 1996 ). TG , thapsigargin.

    Techniques Used: Incubation, Isolation, Agarose Gel Electrophoresis, Northern Blot, SDS Page, Western Blot

    The kinetics of activation of the calreticulin promoter by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and β-galactosidase) were incubated with 7 μM A23187, 100 nM thapsigargin, or DMSO (control cells) for the times indicated. At different time points, cells were either harvested ( open bars ) or washed with PBS and then incubated in drug-free media to a total of 16 h of incubation ( cross-hatched bars ). CAT protein levels and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four separate experiments performed in triplicate.
    Figure Legend Snippet: The kinetics of activation of the calreticulin promoter by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and β-galactosidase) were incubated with 7 μM A23187, 100 nM thapsigargin, or DMSO (control cells) for the times indicated. At different time points, cells were either harvested ( open bars ) or washed with PBS and then incubated in drug-free media to a total of 16 h of incubation ( cross-hatched bars ). CAT protein levels and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four separate experiments performed in triplicate.

    Techniques Used: Activation Assay, Stable Transfection, Expressing, Incubation, Activity Assay

    9) Product Images from "The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity"

    Article Title: The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00001-12

    Role of intracellular Ca 2+ on transepithelial electrical resistance (TER) in human Caco-2 cells. BAPTA/AM and thapsigargin were applied (A) without alpha-toxin (□, control; ▾, BAPTA/AM; ♦, thapsigargin) or (B) with basolaterally
    Figure Legend Snippet: Role of intracellular Ca 2+ on transepithelial electrical resistance (TER) in human Caco-2 cells. BAPTA/AM and thapsigargin were applied (A) without alpha-toxin (□, control; ▾, BAPTA/AM; ♦, thapsigargin) or (B) with basolaterally

    Techniques Used:

    10) Product Images from "Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion"

    Article Title: Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion

    Journal: Nature cell biology

    doi: 10.1038/ncb2614

    Direct measurements of enhancement in secretion due to Ca2+ and F-actin oscillations a Bar diagram showing that secretion rates are more efficient for cells that oscillate (blue) compared to cells with elevated but non-oscillating Ca2+ levels (red). Addition of thapsigargin or cyotocholasin D to oscillating cells abolish the enhanced secretion without significantly affecting secretion when added to non-oscillating cells. Addition of Jasplakinolide abolished the oscillating cell’s advantage and caused an additional overall reduction in secretion. Addition of DMSO is included as a control. (N-oscillating = 2891, N-non-oscillating = 4642 cells; errorbars are s.e.m. Result of t-tests comparing the exocytosis level of oscillating cells to non-oscillating, secretion before the addition of drug or secretion after addition of DMSO are presented above the bar plot., n.s. not statistically significant, * P-value
    Figure Legend Snippet: Direct measurements of enhancement in secretion due to Ca2+ and F-actin oscillations a Bar diagram showing that secretion rates are more efficient for cells that oscillate (blue) compared to cells with elevated but non-oscillating Ca2+ levels (red). Addition of thapsigargin or cyotocholasin D to oscillating cells abolish the enhanced secretion without significantly affecting secretion when added to non-oscillating cells. Addition of Jasplakinolide abolished the oscillating cell’s advantage and caused an additional overall reduction in secretion. Addition of DMSO is included as a control. (N-oscillating = 2891, N-non-oscillating = 4642 cells; errorbars are s.e.m. Result of t-tests comparing the exocytosis level of oscillating cells to non-oscillating, secretion before the addition of drug or secretion after addition of DMSO are presented above the bar plot., n.s. not statistically significant, * P-value

    Techniques Used:

    Synchronized phase-shifted oscillations of Calcium, PI(4,5)P2, N-WASP, and F-actin Ca2+ oscillations drive cortical F-actin oscillations. a Example of a live-cell 5-channel analysis of Ca2+ (Fura2), PI(4,5)P2, N-WASP and F-actin signals shows synchronized oscillations. Stopping Ca2+ oscillations using the SERCA ER pump inhibitor thapsigargin blocks all oscillations. b Changes in the intensity of the four biosensor in a representative cell. The time is indicated when actin oscillations where inhibited using 4 μM Latrunculin. c Suppressing actin polymerization using cytocholasin D blocked F-actin oscillations but not the other oscillations. Raw data for panels a, b c is presented in Supp Table 1 . d Schematics of the Ca2+ pathway that generates coordinated cortical F-actin oscillations. P-value indicates probability that the lag between sequential biosensors is bigger than zero based on cross-correlation analysis of 83 cycles in 4 cells. e Average normalized cycle intensities of all cycles. Color codes are the same as in panels a and b.
    Figure Legend Snippet: Synchronized phase-shifted oscillations of Calcium, PI(4,5)P2, N-WASP, and F-actin Ca2+ oscillations drive cortical F-actin oscillations. a Example of a live-cell 5-channel analysis of Ca2+ (Fura2), PI(4,5)P2, N-WASP and F-actin signals shows synchronized oscillations. Stopping Ca2+ oscillations using the SERCA ER pump inhibitor thapsigargin blocks all oscillations. b Changes in the intensity of the four biosensor in a representative cell. The time is indicated when actin oscillations where inhibited using 4 μM Latrunculin. c Suppressing actin polymerization using cytocholasin D blocked F-actin oscillations but not the other oscillations. Raw data for panels a, b c is presented in Supp Table 1 . d Schematics of the Ca2+ pathway that generates coordinated cortical F-actin oscillations. P-value indicates probability that the lag between sequential biosensors is bigger than zero based on cross-correlation analysis of 83 cycles in 4 cells. e Average normalized cycle intensities of all cycles. Color codes are the same as in panels a and b.

    Techniques Used:

    11) Product Images from "Modification of STIM1 by O-linked N-Acetylglucosamine (O-GlcNAc) Attenuates Store-operated Calcium Entry in Neonatal Cardiomyocytes *"

    Article Title: Modification of STIM1 by O-linked N-Acetylglucosamine (O-GlcNAc) Attenuates Store-operated Calcium Entry in Neonatal Cardiomyocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.383778

    A , effects of glucosamine and thiamet-G on SOCE. NRVMs were pretreated with or without GlcN or TMG. Left panel : representative fluorescence intensity traces from a group of NRVMs before and after thapsigargin treatment followed by Ca 2+ addition; Right
    Figure Legend Snippet: A , effects of glucosamine and thiamet-G on SOCE. NRVMs were pretreated with or without GlcN or TMG. Left panel : representative fluorescence intensity traces from a group of NRVMs before and after thapsigargin treatment followed by Ca 2+ addition; Right

    Techniques Used: Fluorescence

    STIM1 forms puncta and interacts with Orai1 in NRVMs. A , eYFP-STIM1 infected NRVMs were treated with thapsigargin (TG, 5 μ m ) plus EGTA (2 m m ) to deplete ER/SR Ca 2+ . Left images from before (0 min) and at 1 and 3 min after treatment are shown.
    Figure Legend Snippet: STIM1 forms puncta and interacts with Orai1 in NRVMs. A , eYFP-STIM1 infected NRVMs were treated with thapsigargin (TG, 5 μ m ) plus EGTA (2 m m ) to deplete ER/SR Ca 2+ . Left images from before (0 min) and at 1 and 3 min after treatment are shown.

    Techniques Used: Infection

    STIM1 puncta formation was blunted by glucosamine or thiamet-G treatment. A , fluorescent images of NRVMs with eYFP-STIM1 transfection overnight before (0 min) and 3 min following treatment with thapsigargin (5 μ m ) plus EGTA (2 m m ) to deplete ER/SR
    Figure Legend Snippet: STIM1 puncta formation was blunted by glucosamine or thiamet-G treatment. A , fluorescent images of NRVMs with eYFP-STIM1 transfection overnight before (0 min) and 3 min following treatment with thapsigargin (5 μ m ) plus EGTA (2 m m ) to deplete ER/SR

    Techniques Used: Transfection

    12) Product Images from "PLEKHN1 promotes apoptosis by enhancing Bax-Bak hetro-oligomerization through interaction with Bid in human colon cancer"

    Article Title: PLEKHN1 promotes apoptosis by enhancing Bax-Bak hetro-oligomerization through interaction with Bid in human colon cancer

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-017-0006-5

    PLEK-KO cells reduce TG- or ROS-induced cell death a The image of DIC/DAPI staining. The merged images could be used to determine the proportion of apoptotic cells. b HT-29 cells in the presence of 0.1% DMSO with DAPI staining. b ′ PLEK-KO of control medium. Arrow indicates cell during mitosis (mit) c HT-29 cells in the presence of 1 µM thapsigargin (TG)-treatment for 24 h. Arrowheads indicate fragmented nuclei. c ′ PLEK-KO in 1 µM TG same as c . Less pycnotic cells were observed. d Immunofluorescence using active-caspase-3 antibody/Alexa546 rabbit IgG, FITC-phalloidin, and DAPI. Arrowheads indicate dead cells as measured by the accumulation of active-caspase-3 (depicted in red). d ′ PLEK-KO cells as same as d . e The ratio of cell death after chemical treatments between HT-29 and PLEK-KO cells. The difference of the ratio of apoptosis in TG 24 h and H 2 O 2 24 h were significant (*** P
    Figure Legend Snippet: PLEK-KO cells reduce TG- or ROS-induced cell death a The image of DIC/DAPI staining. The merged images could be used to determine the proportion of apoptotic cells. b HT-29 cells in the presence of 0.1% DMSO with DAPI staining. b ′ PLEK-KO of control medium. Arrow indicates cell during mitosis (mit) c HT-29 cells in the presence of 1 µM thapsigargin (TG)-treatment for 24 h. Arrowheads indicate fragmented nuclei. c ′ PLEK-KO in 1 µM TG same as c . Less pycnotic cells were observed. d Immunofluorescence using active-caspase-3 antibody/Alexa546 rabbit IgG, FITC-phalloidin, and DAPI. Arrowheads indicate dead cells as measured by the accumulation of active-caspase-3 (depicted in red). d ′ PLEK-KO cells as same as d . e The ratio of cell death after chemical treatments between HT-29 and PLEK-KO cells. The difference of the ratio of apoptosis in TG 24 h and H 2 O 2 24 h were significant (*** P

    Techniques Used: Staining, Immunofluorescence

    13) Product Images from "Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties"

    Article Title: Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.21.9456-9469.2004

    STC2 is upregulated by the UPR. (A) N2a and PC12 cells were treated with 2 μg of tunicamycin/ml or 300 nM thapsigargin for indicated periods. Total RNA was subject to RT-PCR analyses with STC1 and STC2 primers. UPR was assessed by the induction of BiP mRNA. (B) Quantitative real-time PCR. Steady-state levels of STC2 and β -actin mRNAs were simultaneously quantified in one reaction by using specific primer pairs and fluorescently labeled internal primers. STC2 values were normalized to β -actin mRNA signal and plotted as the fold increase compared to no treatment control. The data represent means ± the SE from three independent experiments. ✽, P
    Figure Legend Snippet: STC2 is upregulated by the UPR. (A) N2a and PC12 cells were treated with 2 μg of tunicamycin/ml or 300 nM thapsigargin for indicated periods. Total RNA was subject to RT-PCR analyses with STC1 and STC2 primers. UPR was assessed by the induction of BiP mRNA. (B) Quantitative real-time PCR. Steady-state levels of STC2 and β -actin mRNAs were simultaneously quantified in one reaction by using specific primer pairs and fluorescently labeled internal primers. STC2 values were normalized to β -actin mRNA signal and plotted as the fold increase compared to no treatment control. The data represent means ± the SE from three independent experiments. ✽, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Labeling

    siRNA inhibition of STC2 induction accentuates cell death by thapsigargin and oxidative stress. (A) N2a cells were cotransfected with empty pSUPER or pSUPER-STC2 siRNA plasmid, along with pEYFP plasmid. Two days after transfection, cells were treated or not treated with 150 nM thapsigargin (Tg), 100 μM H 2 O 2 , 2 μg of tunicamycin/ml (Tm), or 250 nM STS for 16 h. Western blot analysis of STC2, BiP, and β-actin levels. Note the marked reduction in STC2 levels in STC2 siRNA transfected cells. (B) Transfected N2a cells grown on coverslips were washed in PBS and stained with EthD-1 to stain dead cells. Fluorescence images were acquired, and live green cells devoid of EthD-1staining were counted by using MetaMorph software. On the left, the average number of live transfected cells per field in untreated cultures (non) were plotted. On the right, a graph shows the percentage of cell survival (means ± the SE) in cultures treated with various agents (above), normalized to untreated controls in each case, calculated from four independent experiments. ✽, P
    Figure Legend Snippet: siRNA inhibition of STC2 induction accentuates cell death by thapsigargin and oxidative stress. (A) N2a cells were cotransfected with empty pSUPER or pSUPER-STC2 siRNA plasmid, along with pEYFP plasmid. Two days after transfection, cells were treated or not treated with 150 nM thapsigargin (Tg), 100 μM H 2 O 2 , 2 μg of tunicamycin/ml (Tm), or 250 nM STS for 16 h. Western blot analysis of STC2, BiP, and β-actin levels. Note the marked reduction in STC2 levels in STC2 siRNA transfected cells. (B) Transfected N2a cells grown on coverslips were washed in PBS and stained with EthD-1 to stain dead cells. Fluorescence images were acquired, and live green cells devoid of EthD-1staining were counted by using MetaMorph software. On the left, the average number of live transfected cells per field in untreated cultures (non) were plotted. On the right, a graph shows the percentage of cell survival (means ± the SE) in cultures treated with various agents (above), normalized to untreated controls in each case, calculated from four independent experiments. ✽, P

    Techniques Used: Inhibition, Plasmid Preparation, Transfection, Western Blot, Staining, Ethidium Homodimer Assay, Fluorescence, Software

    UPR-mediated expression and secretion of STC2. (A) Specificity of STC2 antiserum. N2a cells were transfected with empty vector or plasmids encoding C-terminally tagged STC1 or untagged STC2. Aliquots of detergent lysates and conditioned media were analyzed by immunoblotting with STC2 antiserum or CT11 (tag) antibody. Note that STC2 antibody does not cross-react with STC1. (B) N2a cells were treated with tunicamycin or thapsigargin for the indicated periods, and detergent lysates were analyzed by blotting with STC2, BiP, CHOP, and β-actin antibodies. Secreted STC2 were immunoprecipitated with STC2 antiserum and then analyzed by immunoblotting with the same antibody. Note the UPR-mediated induction and secretion of STC2.
    Figure Legend Snippet: UPR-mediated expression and secretion of STC2. (A) Specificity of STC2 antiserum. N2a cells were transfected with empty vector or plasmids encoding C-terminally tagged STC1 or untagged STC2. Aliquots of detergent lysates and conditioned media were analyzed by immunoblotting with STC2 antiserum or CT11 (tag) antibody. Note that STC2 antibody does not cross-react with STC1. (B) N2a cells were treated with tunicamycin or thapsigargin for the indicated periods, and detergent lysates were analyzed by blotting with STC2, BiP, CHOP, and β-actin antibodies. Secreted STC2 were immunoprecipitated with STC2 antiserum and then analyzed by immunoblotting with the same antibody. Note the UPR-mediated induction and secretion of STC2.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

    14) Product Images from "LncRNA GOLGA2P10 is induced by PERK/ATF4/CHOP signaling and protects tumor cells from ER stress-induced apoptosis by regulating Bcl-2 family members"

    Article Title: LncRNA GOLGA2P10 is induced by PERK/ATF4/CHOP signaling and protects tumor cells from ER stress-induced apoptosis by regulating Bcl-2 family members

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2469-1

    GOLGA2P10 is upregulated in HCC tissues and upon ER stress. a Tunicamycin or thapsigargin treatment induced GOLGA2P10 expression. Hepatoma cell lines, including MHCC-97H, QGY-7703, and SK-HEP-1, were treated with DMSO (vehicle control), tunicamycin or thapsigargin for 16 h before qPCR analysis. b Tunicamycin treatment increased GOLGA2P10 level in a dose-dependent manner. MHCC-97H cells were treated with DMSO or the indicated doses of tunicamycin for 16 h before qPCR analysis. For a , b , the mean value of DMSO-treated cells was set as relative level 1. Data are shown as mean ± SEM of three independent experiments. Tm tunicamycin, Tg thapsigargin. c GOLGA2P10 was upregulated in HCC tissues. GOLGA2P10 levels were detected in 31 paired HCC (T) and adjacent non-tumor liver tissues (N) by qPCR analysis. The mean value of adjacent non-tumor liver tissues was set as relative level 1. d Kaplan–Meier plots revealed a correlation between high GOLGA2P10 expression and short recurrence-free survival. GOLGA2P10 levels were analyzed in HCC tissues ( n = 156) by qPCR and the median value was chosen as the cut-off point for separating the GOLGA2P10-low level group ( n = 78) from the GOLGA2P10-high level group ( n = 78). β-actin was used as an internal control. * P
    Figure Legend Snippet: GOLGA2P10 is upregulated in HCC tissues and upon ER stress. a Tunicamycin or thapsigargin treatment induced GOLGA2P10 expression. Hepatoma cell lines, including MHCC-97H, QGY-7703, and SK-HEP-1, were treated with DMSO (vehicle control), tunicamycin or thapsigargin for 16 h before qPCR analysis. b Tunicamycin treatment increased GOLGA2P10 level in a dose-dependent manner. MHCC-97H cells were treated with DMSO or the indicated doses of tunicamycin for 16 h before qPCR analysis. For a , b , the mean value of DMSO-treated cells was set as relative level 1. Data are shown as mean ± SEM of three independent experiments. Tm tunicamycin, Tg thapsigargin. c GOLGA2P10 was upregulated in HCC tissues. GOLGA2P10 levels were detected in 31 paired HCC (T) and adjacent non-tumor liver tissues (N) by qPCR analysis. The mean value of adjacent non-tumor liver tissues was set as relative level 1. d Kaplan–Meier plots revealed a correlation between high GOLGA2P10 expression and short recurrence-free survival. GOLGA2P10 levels were analyzed in HCC tissues ( n = 156) by qPCR and the median value was chosen as the cut-off point for separating the GOLGA2P10-low level group ( n = 78) from the GOLGA2P10-high level group ( n = 78). β-actin was used as an internal control. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    ER stress stimulates GOLGA2P10 transcription via the PERK/ATF4/CHOP signaling pathway. a Treatment with actinomycin D abrogated tunicamycin-induced GOLGA2P10 expression. MHCC-97H cells treated with DMSO or tunicamycin were incubated without or with actinomycin D (actD) for 13 h before qPCR analysis. b , c Silencing of PERK or ATF4 diminished tunicamycin-induced GOLGA2P10 expression in MHCC-97H cells. d , e CHOP knockdown abrogated tunicamycin- and thapsigargin-induced GOLGA2P10 expression. For b – e , hepatoma cells were reversely transfected with the indicated RNA duplexes for 36 h, then cultured with DMSO ( b – e ), tunicamycin ( b – d ), or thapsigargin ( e ) for an additional 16 h before qPCR analysis. iMAX, cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. NC, negative control for siRNAs. For a – e , data are shown as mean ± SEM of three independent experiments. f CHOP was upregulated in HCC tissues. The mRNA level of CHOP was detected in 31 paired HCC (T) and adjacent non-tumor liver tissues (N) by qPCR analysis. The mean value of adjacent non-tumor liver tissues was set as relative level 1. g HCCs with CHOP elevation displayed higher GOLGA2P10 expression. The levels of CHOP or GOLGA2P10 in HCC tissue relative to that in adjacent non-tumor tissue (T/N), based on data from Figs. 2f and 1c , were used for analysis. T/N = 1.5 was chosen as the cut-off point for separating the tumors without (−) CHOP-upregulation ( n = 19) from those with (+) CHOP-upregulation ( n = 12). β-actin was used as an internal control. * P
    Figure Legend Snippet: ER stress stimulates GOLGA2P10 transcription via the PERK/ATF4/CHOP signaling pathway. a Treatment with actinomycin D abrogated tunicamycin-induced GOLGA2P10 expression. MHCC-97H cells treated with DMSO or tunicamycin were incubated without or with actinomycin D (actD) for 13 h before qPCR analysis. b , c Silencing of PERK or ATF4 diminished tunicamycin-induced GOLGA2P10 expression in MHCC-97H cells. d , e CHOP knockdown abrogated tunicamycin- and thapsigargin-induced GOLGA2P10 expression. For b – e , hepatoma cells were reversely transfected with the indicated RNA duplexes for 36 h, then cultured with DMSO ( b – e ), tunicamycin ( b – d ), or thapsigargin ( e ) for an additional 16 h before qPCR analysis. iMAX, cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. NC, negative control for siRNAs. For a – e , data are shown as mean ± SEM of three independent experiments. f CHOP was upregulated in HCC tissues. The mRNA level of CHOP was detected in 31 paired HCC (T) and adjacent non-tumor liver tissues (N) by qPCR analysis. The mean value of adjacent non-tumor liver tissues was set as relative level 1. g HCCs with CHOP elevation displayed higher GOLGA2P10 expression. The levels of CHOP or GOLGA2P10 in HCC tissue relative to that in adjacent non-tumor tissue (T/N), based on data from Figs. 2f and 1c , were used for analysis. T/N = 1.5 was chosen as the cut-off point for separating the tumors without (−) CHOP-upregulation ( n = 19) from those with (+) CHOP-upregulation ( n = 12). β-actin was used as an internal control. * P

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Transfection, Cell Culture, Negative Control

    GOLGA2P10 confers tumor cells with resistance to ER stress-induced apoptosis. a – c Silencing of GOLGA2P10 sensitized hepatoma cells to tunicamycin-induced apoptosis. Tumor cells transfected with the indicated RNA duplexes were treated with DMSO or tunicamycin for 48 (MHCC-97H), 28 (QGY-7703), or 52 h (SK-HEP-1), followed by Annexin V/PI staining and flow cytometry analysis ( a ), DAPI staining and nuclear morphological examination ( b ), or Western blotting analysis for active casepase-3 ( c ). d Inhibition of GOLGA2P10 sensitized hepatoma cells to thapsigargin-induced cell death. Hepatoma cells transfected with the indicated RNA duplexes were treated with DMSO or thapsigargin for 48 (MHCC-97H), 28 (QGY-7703), or 52 h (SK-HEP-1), followed by DAPI staining and nuclear morphological examination. e Ectopic expression of GOLGA2P10 attenuated tunicamycin-induced cell death. Hepatoma cells with stable overexpression of GOLGA2P10 (P10) or control (Ctrl) vector were treated with DMSO or tunicamycin for 36 (QGY-7703) or 60 h (SK-HEP-1) before DAPI staining. f GOLGA2P10 knockdown promoted the release of cytochrome C from mitochondria to the cytosol. QGY-7703 cells transfected with the indicated RNA duplexes were incubated with DMSO (−) or tunicamycin (+) for 28 h, followed by Western blotting analysis for cytochrome C (Cyto C) levels in the cytoplasm and in the whole cell extract. β-actin was used as an internal control. iMAX, cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. NC, negative control for siRNAs. siP10-1 and siP10-2, siRNAs targeting different regions of GOLGA2P10. For a , b , d , e , data are shown as mean ± SEM of three independent experiments. For Western blotting in c , f , two independent experiments were performed with similar results. * P
    Figure Legend Snippet: GOLGA2P10 confers tumor cells with resistance to ER stress-induced apoptosis. a – c Silencing of GOLGA2P10 sensitized hepatoma cells to tunicamycin-induced apoptosis. Tumor cells transfected with the indicated RNA duplexes were treated with DMSO or tunicamycin for 48 (MHCC-97H), 28 (QGY-7703), or 52 h (SK-HEP-1), followed by Annexin V/PI staining and flow cytometry analysis ( a ), DAPI staining and nuclear morphological examination ( b ), or Western blotting analysis for active casepase-3 ( c ). d Inhibition of GOLGA2P10 sensitized hepatoma cells to thapsigargin-induced cell death. Hepatoma cells transfected with the indicated RNA duplexes were treated with DMSO or thapsigargin for 48 (MHCC-97H), 28 (QGY-7703), or 52 h (SK-HEP-1), followed by DAPI staining and nuclear morphological examination. e Ectopic expression of GOLGA2P10 attenuated tunicamycin-induced cell death. Hepatoma cells with stable overexpression of GOLGA2P10 (P10) or control (Ctrl) vector were treated with DMSO or tunicamycin for 36 (QGY-7703) or 60 h (SK-HEP-1) before DAPI staining. f GOLGA2P10 knockdown promoted the release of cytochrome C from mitochondria to the cytosol. QGY-7703 cells transfected with the indicated RNA duplexes were incubated with DMSO (−) or tunicamycin (+) for 28 h, followed by Western blotting analysis for cytochrome C (Cyto C) levels in the cytoplasm and in the whole cell extract. β-actin was used as an internal control. iMAX, cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. NC, negative control for siRNAs. siP10-1 and siP10-2, siRNAs targeting different regions of GOLGA2P10. For a , b , d , e , data are shown as mean ± SEM of three independent experiments. For Western blotting in c , f , two independent experiments were performed with similar results. * P

    Techniques Used: Transfection, Staining, Flow Cytometry, Western Blot, Inhibition, Expressing, Over Expression, Plasmid Preparation, Incubation, Negative Control

    15) Product Images from "Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development"

    Article Title: Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development

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

    doi:

    ( A ) Effect of high K + on [Ca 2+ ] i oscillations and contractions observed with CLSM in a single stage-2 cardiomyocyte. Depolarization resulted in a transient rise of [Ca 2+ ] i , presumably because of the activation of VDCC channels and in an alteration of the amplitude and frequency of oscillations. The inset shows cell contractions time-locked with [Ca 2+ ] i oscillations during superfusion with high K + . Cell membrane depolarization resulted in an initial discontinuation of contractions, which resumed with the decline of [Ca 2+ ] i. F/Fo indicates the ratio between the actual and the initial intensity of the fluorescent dye. Grey-levels represent the intensity of the transmitted light, which varied with cell contraction. ( B – D ) Effect of different pharmacological agents on 340/380 ratios in fura-2AM-loaded spontaneously beating stage-2 cells. ( B ) The VDCC blocker nisoldipine (2 μM) did not impair [Ca 2+ ] i oscillations but lead to a lowering of steady-state [Ca 2+ ] i levels. ( C ) Similarly, Ni 2+ (2 mM), known to block the Na + -Ca 2+ exchanger, did not interrupt [Ca 2+ ] i oscillations. ( D ) The Ca 2+ -ATPase inhibitor thapsigargin (100 nM) led to an abrupt halt of the [Ca 2+ ] i oscillations and a concomitant increase of the resting [Ca 2+ ] i .
    Figure Legend Snippet: ( A ) Effect of high K + on [Ca 2+ ] i oscillations and contractions observed with CLSM in a single stage-2 cardiomyocyte. Depolarization resulted in a transient rise of [Ca 2+ ] i , presumably because of the activation of VDCC channels and in an alteration of the amplitude and frequency of oscillations. The inset shows cell contractions time-locked with [Ca 2+ ] i oscillations during superfusion with high K + . Cell membrane depolarization resulted in an initial discontinuation of contractions, which resumed with the decline of [Ca 2+ ] i. F/Fo indicates the ratio between the actual and the initial intensity of the fluorescent dye. Grey-levels represent the intensity of the transmitted light, which varied with cell contraction. ( B – D ) Effect of different pharmacological agents on 340/380 ratios in fura-2AM-loaded spontaneously beating stage-2 cells. ( B ) The VDCC blocker nisoldipine (2 μM) did not impair [Ca 2+ ] i oscillations but lead to a lowering of steady-state [Ca 2+ ] i levels. ( C ) Similarly, Ni 2+ (2 mM), known to block the Na + -Ca 2+ exchanger, did not interrupt [Ca 2+ ] i oscillations. ( D ) The Ca 2+ -ATPase inhibitor thapsigargin (100 nM) led to an abrupt halt of the [Ca 2+ ] i oscillations and a concomitant increase of the resting [Ca 2+ ] i .

    Techniques Used: Confocal Laser Scanning Microscopy, Activation Assay, Blocking Assay

    Pharmacological characterization of the APs and membrane potential fluctuations in spontaneously beating stage-1 and -2 cells measured under current clamp. ( A ) Superfusion of 140 mM K + led to depolarization of the resting potential close to 0 mV and halted both APs and small membrane potential fluctuations. ( B ) Superfusion of the VDCC blocker nisoldipine (50 nM) interrupted the APs but not the small membrane potential fluctuations. ( C ) Application of the sarcoplasmic ATPase inhibitor thapsigargin (100 nM) to a stage-1 cell blocked both APs and membrane potential fluctuations and led to a depolarization of the resting potential.
    Figure Legend Snippet: Pharmacological characterization of the APs and membrane potential fluctuations in spontaneously beating stage-1 and -2 cells measured under current clamp. ( A ) Superfusion of 140 mM K + led to depolarization of the resting potential close to 0 mV and halted both APs and small membrane potential fluctuations. ( B ) Superfusion of the VDCC blocker nisoldipine (50 nM) interrupted the APs but not the small membrane potential fluctuations. ( C ) Application of the sarcoplasmic ATPase inhibitor thapsigargin (100 nM) to a stage-1 cell blocked both APs and membrane potential fluctuations and led to a depolarization of the resting potential.

    Techniques Used:

    16) Product Images from "Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia"

    Article Title: Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia

    Journal: Mucosal immunology

    doi: 10.1038/mi.2014.24

    MRSA induction of IL-16 release is calcium and calpain dependent in THP-1s Calcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p
    Figure Legend Snippet: MRSA induction of IL-16 release is calcium and calpain dependent in THP-1s Calcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p

    Techniques Used: Fluorescence, Activation Assay, Inverted Microscopy, Positive Control, Infection

    17) Product Images from "NF-?B Inhibitors from Brucea javanica Exhibiting Intracellular Effects on Reactive Oxygen Species"

    Article Title: NF-?B Inhibitors from Brucea javanica Exhibiting Intracellular Effects on Reactive Oxygen Species

    Journal: Anticancer research

    doi:

    Effects on NFAT nuclear translocation. Panels left to right: a: Chrysoeriol ( 8 ); b: thapsigargin control; c: PBS control; and d: rocaglamide control. All treatments were performed in duplicate and repeated in at least an additional independent experiment.
    Figure Legend Snippet: Effects on NFAT nuclear translocation. Panels left to right: a: Chrysoeriol ( 8 ); b: thapsigargin control; c: PBS control; and d: rocaglamide control. All treatments were performed in duplicate and repeated in at least an additional independent experiment.

    Techniques Used: Translocation Assay

    18) Product Images from "Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease"

    Article Title: Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-8-45

    Cyclosporine A prevented Reactive Oxygen Species (ROS) production and mMTP opening induced by thapsigargin in mutant huntingtin cells. A , representative fluorescence images of 2,7-dichlorofluorescein indicating the levels of ROS striatal cells challenged with 1 μM thapsigargin (Th) for 1 h, CsA for 2 h and both. Increased levels of ROS in striatal cells treated with thapsigargin (A and B) were prevented by pretreatment with CsA. Bar =10 μm. B , Quantitative ROS levels. Data are the mean ± S.E.M. of 3 independent experiments. *, p
    Figure Legend Snippet: Cyclosporine A prevented Reactive Oxygen Species (ROS) production and mMTP opening induced by thapsigargin in mutant huntingtin cells. A , representative fluorescence images of 2,7-dichlorofluorescein indicating the levels of ROS striatal cells challenged with 1 μM thapsigargin (Th) for 1 h, CsA for 2 h and both. Increased levels of ROS in striatal cells treated with thapsigargin (A and B) were prevented by pretreatment with CsA. Bar =10 μm. B , Quantitative ROS levels. Data are the mean ± S.E.M. of 3 independent experiments. *, p

    Techniques Used: Mutagenesis, Fluorescence

    Mitochondrial impairment induced by thapsigargin was prevented by cyclosporine A in neurons that expressed mutant huntingtin. A , representative fluorescence images of cortical neurons loaded with calcein blue AM (Calcein B) and transfected with GFP alone, Q25-GFP (normal huntingtin), or Q104-GFP (mutant huntingtin). Normal and mutant huntingtin expression is indicated by GFP fluorescence intensity (white arrows) and calcein blue staining reveals neuronal morphology. Bar = 10 μm. B , transfected neurons were loaded with MitoRed for determination of mitochondrial potential. Representative trends show that the expression of Q104-GFP significantly reduced mitochondrial potential during treatment with 1 μM thapsigargin (Th). C , quantitation of mitochondrial potential levels showed significant mitochondrial damage in neurons that expressed Q104-GFP. Data correspond to the mean ± S.E.M. of 4 independent experiments. *, p
    Figure Legend Snippet: Mitochondrial impairment induced by thapsigargin was prevented by cyclosporine A in neurons that expressed mutant huntingtin. A , representative fluorescence images of cortical neurons loaded with calcein blue AM (Calcein B) and transfected with GFP alone, Q25-GFP (normal huntingtin), or Q104-GFP (mutant huntingtin). Normal and mutant huntingtin expression is indicated by GFP fluorescence intensity (white arrows) and calcein blue staining reveals neuronal morphology. Bar = 10 μm. B , transfected neurons were loaded with MitoRed for determination of mitochondrial potential. Representative trends show that the expression of Q104-GFP significantly reduced mitochondrial potential during treatment with 1 μM thapsigargin (Th). C , quantitation of mitochondrial potential levels showed significant mitochondrial damage in neurons that expressed Q104-GFP. Data correspond to the mean ± S.E.M. of 4 independent experiments. *, p

    Techniques Used: Mutagenesis, Fluorescence, Transfection, Expressing, Staining, Quantitation Assay

    Treatment with Cyclosporine A prevents mitochondrial injury induced by thapsigargin in mutant HD cells. A , live confocal images of MitoRed, an indicator of mitochondrial potential, in striatal cells, untreated and treated with 1 μM thapsigargin (Th) for 30 min. Thapsigargin induces a significant decrease in the mitochondrial potential in mutant cells (see white arrows). Bar represents 10 μm. B , images show that CsA protects mutant huntingtin cells exposed to thapsigargin from mitochondrial impairment. Bar represents 10 μm. C , quantification of MitoRed intensities as relative units (ΔF/F 0 ) in striatal cells treated for 30 min with experimental conditions as indicated. Data are the mean ± S.E.M. of 4 independent experiments. *, p
    Figure Legend Snippet: Treatment with Cyclosporine A prevents mitochondrial injury induced by thapsigargin in mutant HD cells. A , live confocal images of MitoRed, an indicator of mitochondrial potential, in striatal cells, untreated and treated with 1 μM thapsigargin (Th) for 30 min. Thapsigargin induces a significant decrease in the mitochondrial potential in mutant cells (see white arrows). Bar represents 10 μm. B , images show that CsA protects mutant huntingtin cells exposed to thapsigargin from mitochondrial impairment. Bar represents 10 μm. C , quantification of MitoRed intensities as relative units (ΔF/F 0 ) in striatal cells treated for 30 min with experimental conditions as indicated. Data are the mean ± S.E.M. of 4 independent experiments. *, p

    Techniques Used: Mutagenesis

    Effects of FK-506 on mitochondria impairment induced by thapsigargin in mutant huntingtin cells. A , wild type and mutant cells were incubated with 500 nM FK-506, an inhibitor of calcineurin activity, for 2 h prior to mitochondrial potential determinations using MitoRed. Striatal cells loaded with MitoRed were exposed to 1 μM thapsigargin (Th) for 30 min, and mitochondrial potential was evaluated using confocal microscopy. Treatment with thapsigargin reduced mitochondrial potential levels in mutant striatal cells pre-treated with FK-506, indicating that calcineurin activity was not playing a role in mitochondrial impairment induced by calcium stress in mutant huntingtin cells. B , quantitation of MitoRed intensities, which represents mitochondrial potential levels, as relative units at 30 min (ΔF/F 0 ) in striatal cells treated with conditions indicated. Data are expressed as the mean ± S.E.M. of 4 independent experiments. *, p
    Figure Legend Snippet: Effects of FK-506 on mitochondria impairment induced by thapsigargin in mutant huntingtin cells. A , wild type and mutant cells were incubated with 500 nM FK-506, an inhibitor of calcineurin activity, for 2 h prior to mitochondrial potential determinations using MitoRed. Striatal cells loaded with MitoRed were exposed to 1 μM thapsigargin (Th) for 30 min, and mitochondrial potential was evaluated using confocal microscopy. Treatment with thapsigargin reduced mitochondrial potential levels in mutant striatal cells pre-treated with FK-506, indicating that calcineurin activity was not playing a role in mitochondrial impairment induced by calcium stress in mutant huntingtin cells. B , quantitation of MitoRed intensities, which represents mitochondrial potential levels, as relative units at 30 min (ΔF/F 0 ) in striatal cells treated with conditions indicated. Data are expressed as the mean ± S.E.M. of 4 independent experiments. *, p

    Techniques Used: Mutagenesis, Incubation, Activity Assay, Confocal Microscopy, Quantitation Assay

    Treatment with Cyclosporine A improved mitochondrial calcium uptake in mutant huntingtin cells exposed to calcium overload. A , striatal cells were loaded with Rhod2 AM dye to determine mitochondrial calcium levels in cells exposed to 1 μM thapsigargin (Th) during 30 min. Thapsigargin treatment severely decreased mitochondrial calcium uptake. Striatal cells were incubated for 2 h with 0.5 μM Cyclosporine A (CsA) prior to determination of mitochondrial calcium levels in response to thapsigargin treatment. CsA prevented the mitochondrial calcium decrease induced by thapsigargin in the mutant cells. B , quantification of mitochondrial calcium levels from 4 independent experiments obtained from cells exposed to thapsigargin for 30 min. *, p
    Figure Legend Snippet: Treatment with Cyclosporine A improved mitochondrial calcium uptake in mutant huntingtin cells exposed to calcium overload. A , striatal cells were loaded with Rhod2 AM dye to determine mitochondrial calcium levels in cells exposed to 1 μM thapsigargin (Th) during 30 min. Thapsigargin treatment severely decreased mitochondrial calcium uptake. Striatal cells were incubated for 2 h with 0.5 μM Cyclosporine A (CsA) prior to determination of mitochondrial calcium levels in response to thapsigargin treatment. CsA prevented the mitochondrial calcium decrease induced by thapsigargin in the mutant cells. B , quantification of mitochondrial calcium levels from 4 independent experiments obtained from cells exposed to thapsigargin for 30 min. *, p

    Techniques Used: Mutagenesis, Incubation

    Mitochondrial fragmentation induced by calcium overload was prevented by inhibition of mPTP in mutant huntingtin cells. A , striatal cells transfected with Mito-GFP protein were treated with 1 μM thapsigargin (Th) during 60 min before live fluorescence images were taken. Images were acquired from full resolution images obtained with 63X objective in a Zeiss Axiovert LED fluorescence microscope. Thapsigargin treatment induced a significant fragmentation of mitochondria in mutant compared with wild type cells. Bar = 10 μm. B , magnification of boxed regions from A to emphasize differences of mitochondrial morphology in striatal cells. Bar = 5 μm. C , striatal cells transfected with Mito-GFP protein were treated with thapsigargin (Th) plus cyclosporine A (CsA) during 60 min and live fluorescence images were obtained. Images were acquired from full resolution images obtained with 63X objective in a Zeiss Axiovert LED fluorescence microscope. Thapsigargin treatment induced significant fragmentation of mitochondria in mutant compared with wild type cells. Mitochondria fragmentation produced by calcium stress was abolished by mPTP closure induced by 0.5 μM CsA treatment for 2 h. Bar = 10 μm. D , magnification of boxed regions from A to emphasize differences of mitochondrial morphology in striatal cells. Bar = 5 μm. E , quantitation of mitochondrial length of 4 independent experiments showed significant mitochondria fragmentation in mutant cells exposed to thapsigargin and this effect was prevented by co-treatment with CsA. Data correspond to the mean ± S.E.M. of 4 independent experiments. * p
    Figure Legend Snippet: Mitochondrial fragmentation induced by calcium overload was prevented by inhibition of mPTP in mutant huntingtin cells. A , striatal cells transfected with Mito-GFP protein were treated with 1 μM thapsigargin (Th) during 60 min before live fluorescence images were taken. Images were acquired from full resolution images obtained with 63X objective in a Zeiss Axiovert LED fluorescence microscope. Thapsigargin treatment induced a significant fragmentation of mitochondria in mutant compared with wild type cells. Bar = 10 μm. B , magnification of boxed regions from A to emphasize differences of mitochondrial morphology in striatal cells. Bar = 5 μm. C , striatal cells transfected with Mito-GFP protein were treated with thapsigargin (Th) plus cyclosporine A (CsA) during 60 min and live fluorescence images were obtained. Images were acquired from full resolution images obtained with 63X objective in a Zeiss Axiovert LED fluorescence microscope. Thapsigargin treatment induced significant fragmentation of mitochondria in mutant compared with wild type cells. Mitochondria fragmentation produced by calcium stress was abolished by mPTP closure induced by 0.5 μM CsA treatment for 2 h. Bar = 10 μm. D , magnification of boxed regions from A to emphasize differences of mitochondrial morphology in striatal cells. Bar = 5 μm. E , quantitation of mitochondrial length of 4 independent experiments showed significant mitochondria fragmentation in mutant cells exposed to thapsigargin and this effect was prevented by co-treatment with CsA. Data correspond to the mean ± S.E.M. of 4 independent experiments. * p

    Techniques Used: Inhibition, Mutagenesis, Transfection, Fluorescence, Microscopy, Produced, Quantitation Assay

    Effect of cell depolarization on calcium regulation in striatal cells. A, B striatal cells were loaded with Fluo3 AM and cytosolic calcium was measured using confocal microscopy. Fluo3 fluorescence changes were measured after cells were treated with 60 mM KCl for 30 min. Fluo3 intensity variations represent changes in cytosolic calcium levels and are expressed using the pseudo ratio ΔF/F 0 (see Methods). B , quantitative analysis of 4 independent experiments of striatal cells treated with 60 mM KCl and 1 μM thapsigargin. No differences in cytosolic calcium peak were observed when wild type and mutant cells were compared. Data correspond to the mean ± S.E.M. of four independent experiments. * p
    Figure Legend Snippet: Effect of cell depolarization on calcium regulation in striatal cells. A, B striatal cells were loaded with Fluo3 AM and cytosolic calcium was measured using confocal microscopy. Fluo3 fluorescence changes were measured after cells were treated with 60 mM KCl for 30 min. Fluo3 intensity variations represent changes in cytosolic calcium levels and are expressed using the pseudo ratio ΔF/F 0 (see Methods). B , quantitative analysis of 4 independent experiments of striatal cells treated with 60 mM KCl and 1 μM thapsigargin. No differences in cytosolic calcium peak were observed when wild type and mutant cells were compared. Data correspond to the mean ± S.E.M. of four independent experiments. * p

    Techniques Used: Confocal Microscopy, Fluorescence, Mutagenesis

    19) Product Images from "Melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in leukocytes from elderly humans"

    Article Title: Melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in leukocytes from elderly humans

    Journal: Age

    doi: 10.1007/s11357-010-9194-0

    Caspase activation is reversed by melatonin in aged leukocytes. Cells from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ) or 10 nM fMLP for 1 h to check caspase-9 ( a and c ) and caspase-3 ( b and d ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-increase over the pre-treatment level (experimental/control). Asterisks P
    Figure Legend Snippet: Caspase activation is reversed by melatonin in aged leukocytes. Cells from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ) or 10 nM fMLP for 1 h to check caspase-9 ( a and c ) and caspase-3 ( b and d ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-increase over the pre-treatment level (experimental/control). Asterisks P

    Techniques Used: Activation Assay, Incubation

    Effects of melatonin on PS exposure in aged leukocytes. Neutrophils ( a ) and lymphocytes ( b ) from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-increase over the pre-treatment level (experimental/control). Asterisks P
    Figure Legend Snippet: Effects of melatonin on PS exposure in aged leukocytes. Neutrophils ( a ) and lymphocytes ( b ) from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-increase over the pre-treatment level (experimental/control). Asterisks P

    Techniques Used: Incubation

    Melatonin counteracts the mitochondrial membrane depolarization induced by calcium mobilizing agents in aged leukocytes. Neutrophils ( a ) and lymphocytes ( b ) from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-change over the pre-treatment level (experimental/control). Asterisks P
    Figure Legend Snippet: Melatonin counteracts the mitochondrial membrane depolarization induced by calcium mobilizing agents in aged leukocytes. Neutrophils ( a ) and lymphocytes ( b ) from both young ( black bars ) and elderly ( grey bars ) individuals were pre-incubated with 1 μM melatonin ( Mel ) or the vehicle for 1 h, and then stimulated with 1 μM thapsigargin ( TG ” section. Values are presented as means ± SEM of six separate experiments and expressed as fold-change over the pre-treatment level (experimental/control). Asterisks P

    Techniques Used: Incubation

    20) Product Images from "Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways *"

    Article Title: Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.004762

    Ca 2+ is necessary but not sufficient by itself for agonist-evoked ATP release. A , cells were preincubated for 20 min with vehicle ( Ctrl ), 1 μ m thapsigargin ( Tg ), or 10 μ m BAPTA-AM ( BAPTA ), and ATP concentrations were measured off-line,
    Figure Legend Snippet: Ca 2+ is necessary but not sufficient by itself for agonist-evoked ATP release. A , cells were preincubated for 20 min with vehicle ( Ctrl ), 1 μ m thapsigargin ( Tg ), or 10 μ m BAPTA-AM ( BAPTA ), and ATP concentrations were measured off-line,

    Techniques Used:

    21) Product Images from "Evidence for an Additive Neurorestorative Effect of Simultaneously Administered CDNF and GDNF in Hemiparkinsonian Rats: Implications for Different Mechanism of Action"

    Article Title: Evidence for an Additive Neurorestorative Effect of Simultaneously Administered CDNF and GDNF in Hemiparkinsonian Rats: Implications for Different Mechanism of Action

    Journal: eNeuro

    doi: 10.1523/ENEURO.0117-16.2017

    Effects of CDNF and GDNF and their combination on ER stress-triggered UPR markers in cultured DA neurons and in rat 6-OHDA model in vivo . A – C , E13 dopamine neurons were cultured 5-7 d with GDNF (50 ng/ml). Then, the cultures were treated with thapsigargin (200 nM) to induce ER stress. CDNF (100 ng/ml), GDNF (50 ng/ml), or their combination was added to the cultures at the same time. The expression levels were normalized to the levels of β-actin in the same samples. CDNF and the combination reduced expression of ATF6 mRNA ( A ). Levels of Xbp1-sp ( B ) and GPR-78 ( C ) were not changed. D- - F , Effect of CDNF and GDNF on ER stress markers in the rat 6-OHDA model. D , Experimental design for ER stress markers analyses. Rats were administered 6-OHDA (20 µg) unilaterally into the left striatum (L). Four weeks later, the rats received a single injection of either vehicle (PBS), CDNF (2.5 µg), GDNF (1 µg), or their combination into the lesioned striatum. Rats were killed 4 h after NTF injection, and striata were dissected for Western blot analyses. E , Injection of CDNF decreases GRP78 protein levels in striata of 6-OHDA-lesioned rats compared with PBS ( p = 0.2) or GDNF treatment ( p
    Figure Legend Snippet: Effects of CDNF and GDNF and their combination on ER stress-triggered UPR markers in cultured DA neurons and in rat 6-OHDA model in vivo . A – C , E13 dopamine neurons were cultured 5-7 d with GDNF (50 ng/ml). Then, the cultures were treated with thapsigargin (200 nM) to induce ER stress. CDNF (100 ng/ml), GDNF (50 ng/ml), or their combination was added to the cultures at the same time. The expression levels were normalized to the levels of β-actin in the same samples. CDNF and the combination reduced expression of ATF6 mRNA ( A ). Levels of Xbp1-sp ( B ) and GPR-78 ( C ) were not changed. D- - F , Effect of CDNF and GDNF on ER stress markers in the rat 6-OHDA model. D , Experimental design for ER stress markers analyses. Rats were administered 6-OHDA (20 µg) unilaterally into the left striatum (L). Four weeks later, the rats received a single injection of either vehicle (PBS), CDNF (2.5 µg), GDNF (1 µg), or their combination into the lesioned striatum. Rats were killed 4 h after NTF injection, and striata were dissected for Western blot analyses. E , Injection of CDNF decreases GRP78 protein levels in striata of 6-OHDA-lesioned rats compared with PBS ( p = 0.2) or GDNF treatment ( p

    Techniques Used: Cell Culture, In Vivo, Expressing, Injection, Western Blot

    22) Product Images from "Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster"

    Article Title: Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151550

    Drug-induced ER stress upregulates DmManf expression. A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of DmManf and Hsc3 were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of Xbp1 , unspliced ( Xbp1 u ) and spliced ( Xbp1 s ). RpL32 was used as a loading control. C–D) qPCR analysis of Hsc3 and Xbp1 expression in DmManf mutant (C) and DmManf overexpressing (D) larvae. Expression of Hsc3 was not altered but Xbp1s mRNA level was increased in response to overexpression of DmManf . The overexpression of DmManf resulted in 165-fold increase in DmManf mRNA level (±23, P
    Figure Legend Snippet: Drug-induced ER stress upregulates DmManf expression. A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of DmManf and Hsc3 were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of Xbp1 , unspliced ( Xbp1 u ) and spliced ( Xbp1 s ). RpL32 was used as a loading control. C–D) qPCR analysis of Hsc3 and Xbp1 expression in DmManf mutant (C) and DmManf overexpressing (D) larvae. Expression of Hsc3 was not altered but Xbp1s mRNA level was increased in response to overexpression of DmManf . The overexpression of DmManf resulted in 165-fold increase in DmManf mRNA level (±23, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis, Over Expression

    23) Product Images from "Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency"

    Article Title: Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency

    Journal: bioRxiv

    doi: 10.1101/864280

    Cav1.4-deficient T lymphocytes exhibit a reduced Ca 2+ flux PBMCs of patients (n=3) and healthy controls (n=8) were stained with Ca 2+ dyes and different T and B lymphocyte-specific antibodies and analyzed by flow cytometry. Thapsigargin was added to cells after 30 seconds of acquisition. The boxplots show the quantified slopes of increasing Ca 2+ concentration for each cell type (A). The flow cytometry kinetics plots show the actual Ca 2+ influx over time (B). Representative of two technical replicates. * p
    Figure Legend Snippet: Cav1.4-deficient T lymphocytes exhibit a reduced Ca 2+ flux PBMCs of patients (n=3) and healthy controls (n=8) were stained with Ca 2+ dyes and different T and B lymphocyte-specific antibodies and analyzed by flow cytometry. Thapsigargin was added to cells after 30 seconds of acquisition. The boxplots show the quantified slopes of increasing Ca 2+ concentration for each cell type (A). The flow cytometry kinetics plots show the actual Ca 2+ influx over time (B). Representative of two technical replicates. * p

    Techniques Used: Staining, Flow Cytometry, Concentration Assay

    24) Product Images from "A polarized Ca2+, diacylglycerol, and STIM1 signaling system regulates directed cell migration"

    Article Title: A polarized Ca2+, diacylglycerol, and STIM1 signaling system regulates directed cell migration

    Journal: Nature cell biology

    doi: 10.1038/ncb2906

    STIM1 is locally activated in the front of migrating cells. (a) HUVEC cells were co-transfected with YFP-STIM1 and the ER-PM junction marker CFP-ER-PM. Confocal images show focal planes at the bottom of the cell. The white arrow marks the direction of migration.YFP-STIM1 was enriched at front ER-PM junctions in migrating cells. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER-PM from front to back in migrating cells. (n =14 cells) (c,d) Similar analysis as in (b) but for cells coexpressing YFP-S1NN and CFP-ER-PM (c) or coexpressing YFP-ER-PM and CFP-ER-PM. A smaller increase in relative S1NN activity was observed in the front (n = 10 cells for S1NN and n = 12 cells for the control group). (e,f) Decreasing luminal ER Ca 2+ levels towards the front of migrating leader cells. (e) Ratio-imaging of a modified luminal ER Ca 2+ FRET probe T1ER (see Methods ) in migrating HUVECs. Adding the SERCA inhibitor thapsigargin (2 μM) and EGTA (3 mM) decreased ER Ca 2+ levels (lower panel). (f) Gradient in luminal ER Ca 2+ measured using the T1ER probe. Note that the lower Ca 2+ levels in the front are still sensitive to EGTA+thapsigargin treatment (n = 79 cells for the control group; n = 49 cells for the thapsigargin + EGTA group).
    Figure Legend Snippet: STIM1 is locally activated in the front of migrating cells. (a) HUVEC cells were co-transfected with YFP-STIM1 and the ER-PM junction marker CFP-ER-PM. Confocal images show focal planes at the bottom of the cell. The white arrow marks the direction of migration.YFP-STIM1 was enriched at front ER-PM junctions in migrating cells. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER-PM from front to back in migrating cells. (n =14 cells) (c,d) Similar analysis as in (b) but for cells coexpressing YFP-S1NN and CFP-ER-PM (c) or coexpressing YFP-ER-PM and CFP-ER-PM. A smaller increase in relative S1NN activity was observed in the front (n = 10 cells for S1NN and n = 12 cells for the control group). (e,f) Decreasing luminal ER Ca 2+ levels towards the front of migrating leader cells. (e) Ratio-imaging of a modified luminal ER Ca 2+ FRET probe T1ER (see Methods ) in migrating HUVECs. Adding the SERCA inhibitor thapsigargin (2 μM) and EGTA (3 mM) decreased ER Ca 2+ levels (lower panel). (f) Gradient in luminal ER Ca 2+ measured using the T1ER probe. Note that the lower Ca 2+ levels in the front are still sensitive to EGTA+thapsigargin treatment (n = 79 cells for the control group; n = 49 cells for the thapsigargin + EGTA group).

    Techniques Used: Transfection, Marker, Migration, Activity Assay, Imaging, Modification

    Higher Ca 2+ pump activity in the front compared to the back generates a gradient of basal [Ca 2+ ] in migrating cells. (a–d) UV flash photolysis experiments confirmed differential Ca 2+ pump activities in migrating HUVECs as shown in Fig. 6c–e . (a) Migrating HUVECs were pre-loaded with Fluo-3/AM and NP-EGTA. Thapsigargin and EGTA were added 10 minutes before imaging to block the activity of SERCA and influx Ca 2+ channels. A UV pulse was used to induce a Ca 2+ spike 1 minute after recording began. (b,c) Ca 2+ pump activities (k) in the front and in the back were calculated based on Fluo-3 measurements following UV photolysis, similar to Fig. 6d,e . (d) Quantification of relative Ca 2+ pump activities in the front and in the back of migrating cells. (n = 41 cells.) (e) Inhibitors of Na + -Ca 2+ exchangers 2,4-DCB or 3,4-DCB both caused a small increase cytosolic Ca 2+ levels as measured by Fura-2. EGTA, Thapsigargin (Th) and BTP2 were used as positive controls. [Ca 2+ ] was normalized using average cytosolic levels in the DMSO group (n = 4 wells for each group). (f) Addition of 10 μM 2,4-DCB or 3,4-DCB did not affect the differential Ca 2+ pump activities. LaCl 3 was used as a positive control (n = 41, 40, 35 42 cells in DMSO, 2,4-DCB, 3,4-DCB and LaCl 3 ). (g) PMCA4 is enriched in the front of migrating cells. HUVEC co-expressing GFP-PMCA4 and the plasma membrane marker tdimer2-lyn were imaged by confocal microscopy. The merged ( left ) and the ratio-image ( right ) indicated enrichment of GFP-PMCA4 in the front (see also Supplementary Fig. 6d ). The white arrow depicts the direction of cell migration. (h) Statistical analysis of the relative spatial distribution of GFP-PMCA4/tdimer2-lyn from the front to the back (n = 13 cells). A Student t test was used for Fig. 7d,f,h . In Fig. 7h, p values were calculated based on the ratio of the sensor / PM ratio in the front 10% region to that in the back 10% region of migrating cells. In Fig. 7d,e,f, Bars are mean ± SEM.
    Figure Legend Snippet: Higher Ca 2+ pump activity in the front compared to the back generates a gradient of basal [Ca 2+ ] in migrating cells. (a–d) UV flash photolysis experiments confirmed differential Ca 2+ pump activities in migrating HUVECs as shown in Fig. 6c–e . (a) Migrating HUVECs were pre-loaded with Fluo-3/AM and NP-EGTA. Thapsigargin and EGTA were added 10 minutes before imaging to block the activity of SERCA and influx Ca 2+ channels. A UV pulse was used to induce a Ca 2+ spike 1 minute after recording began. (b,c) Ca 2+ pump activities (k) in the front and in the back were calculated based on Fluo-3 measurements following UV photolysis, similar to Fig. 6d,e . (d) Quantification of relative Ca 2+ pump activities in the front and in the back of migrating cells. (n = 41 cells.) (e) Inhibitors of Na + -Ca 2+ exchangers 2,4-DCB or 3,4-DCB both caused a small increase cytosolic Ca 2+ levels as measured by Fura-2. EGTA, Thapsigargin (Th) and BTP2 were used as positive controls. [Ca 2+ ] was normalized using average cytosolic levels in the DMSO group (n = 4 wells for each group). (f) Addition of 10 μM 2,4-DCB or 3,4-DCB did not affect the differential Ca 2+ pump activities. LaCl 3 was used as a positive control (n = 41, 40, 35 42 cells in DMSO, 2,4-DCB, 3,4-DCB and LaCl 3 ). (g) PMCA4 is enriched in the front of migrating cells. HUVEC co-expressing GFP-PMCA4 and the plasma membrane marker tdimer2-lyn were imaged by confocal microscopy. The merged ( left ) and the ratio-image ( right ) indicated enrichment of GFP-PMCA4 in the front (see also Supplementary Fig. 6d ). The white arrow depicts the direction of cell migration. (h) Statistical analysis of the relative spatial distribution of GFP-PMCA4/tdimer2-lyn from the front to the back (n = 13 cells). A Student t test was used for Fig. 7d,f,h . In Fig. 7h, p values were calculated based on the ratio of the sensor / PM ratio in the front 10% region to that in the back 10% region of migrating cells. In Fig. 7d,e,f, Bars are mean ± SEM.

    Techniques Used: Activity Assay, Imaging, Blocking Assay, Positive Control, Expressing, Marker, Confocal Microscopy, Migration

    Store-operated Ca 2+ (SOC) influx controls cell migration by regulating cell-matrix adhesion in the front of migrating cells. (a) HUVEC migration into open space was monitored by staining cells with CellMask (see Methods ). Accelerated sheet migration was observed in STIM1-depleted compared to control cells. (b) Comparing changes in the rate of sheet migration and cytosolic Ca 2+ levels in HUVECs treated with siRNAs targeting different Ca 2+ signaling regulators. Average cytosolic [Ca 2+ ] was normalized to the level of cells treated with siCntrl (n = 4 experiments for each siRNA). (c) Reduced single cell migration speed in cells over-expressing YFP-STIM1. Cells expressing YFP-ER were used as control (n ~ 10,000 cells per condition). (d,e) Effects of the ER Ca 2+ pump blocker thapsigargin (d) and the SOC inhibitor BTP2 (e) on cytosolic Ca 2+ levels and on sheet migration speed. Notice that increasing cytosolic Ca 2+ levels by thapsigargin decreased migration speed, and lowering Ca 2+ levels by BTP2 increased migration speed (n = 4 experiments per condition). (f,g) Migrating HUVECs were treated with different concentrations of BTP2 or thapsigargin to reduce or elevate cytosolic Ca 2+ levels. Cells were then fixed and stained with anti-phospho-myosin light chain (pMLC) antibody. (f) pMLC signals were lower when SOC was blocked by BTP2 but higher when ER Ca 2+ pumps were blocked by thapsigargin. CAAX: plasma membrane marker. (g) pMLC levels increased with increasing cytosolic [Ca 2+ ] (n = 123, 134, 127, 126, 123, 117 and 142 cells per condition from left to right). (h,i) Effect of BTP2 treatment on cell-matrix adhesion. Focal adhesion formation was monitored by expressing GFP-paxillin. BTP2 treatment rapidly decreased the intensities of GFP-paxillin puncta in the front of migrating cells, consistent with SOC influx promoting cell-matrix adhesion. Bars are mean ± SEM in Fig. 2b,d,e,g.
    Figure Legend Snippet: Store-operated Ca 2+ (SOC) influx controls cell migration by regulating cell-matrix adhesion in the front of migrating cells. (a) HUVEC migration into open space was monitored by staining cells with CellMask (see Methods ). Accelerated sheet migration was observed in STIM1-depleted compared to control cells. (b) Comparing changes in the rate of sheet migration and cytosolic Ca 2+ levels in HUVECs treated with siRNAs targeting different Ca 2+ signaling regulators. Average cytosolic [Ca 2+ ] was normalized to the level of cells treated with siCntrl (n = 4 experiments for each siRNA). (c) Reduced single cell migration speed in cells over-expressing YFP-STIM1. Cells expressing YFP-ER were used as control (n ~ 10,000 cells per condition). (d,e) Effects of the ER Ca 2+ pump blocker thapsigargin (d) and the SOC inhibitor BTP2 (e) on cytosolic Ca 2+ levels and on sheet migration speed. Notice that increasing cytosolic Ca 2+ levels by thapsigargin decreased migration speed, and lowering Ca 2+ levels by BTP2 increased migration speed (n = 4 experiments per condition). (f,g) Migrating HUVECs were treated with different concentrations of BTP2 or thapsigargin to reduce or elevate cytosolic Ca 2+ levels. Cells were then fixed and stained with anti-phospho-myosin light chain (pMLC) antibody. (f) pMLC signals were lower when SOC was blocked by BTP2 but higher when ER Ca 2+ pumps were blocked by thapsigargin. CAAX: plasma membrane marker. (g) pMLC levels increased with increasing cytosolic [Ca 2+ ] (n = 123, 134, 127, 126, 123, 117 and 142 cells per condition from left to right). (h,i) Effect of BTP2 treatment on cell-matrix adhesion. Focal adhesion formation was monitored by expressing GFP-paxillin. BTP2 treatment rapidly decreased the intensities of GFP-paxillin puncta in the front of migrating cells, consistent with SOC influx promoting cell-matrix adhesion. Bars are mean ± SEM in Fig. 2b,d,e,g.

    Techniques Used: Migration, Staining, Expressing, Marker

    Polarized plasma membrane Ca 2+ pump activity keeps cytosolic Ca 2+ low in the front. (a) Basal cytosolic Ca 2+ levels (measured using Fura-2) in the front were about 50% of the levels in the back of migrating cells (n = 14 cells). Local [Ca 2+ ] was normalized to the average cytosolic level. Cytosolic measurements using GCaMP6s-CAAX are shown in Fig. 1 i,j . (b) High Fura-2 increased the diffusion speed of Ca 2+ , so as to decrease the Ca 2+ gradient between the front and back of migrating cells. Bars are mean ± SEM (n = 160, 160, 80, 160 and 80 cells from left to right for each group). (c) Addition of the PMCA inhibitors Caloxin (200 μM) and La 3+ (200 mM) significantly decreased cytosolic Ca 2+ gradients in migrating cells. Bars are mean ± SEM (n = 47 cells per condition). (d–g) Cytosolic Ca 2+ is transported out of the cell faster in the front than in the back. (d) Addition of thapsigargin, SOC inhibitor BTP2, and Ca 2+ chelator EGTA to migrating HUVECs caused a transient increase in cytosolic Ca 2+ . Local extrusion pump rates were measured as a function of the Ca 2+ level in the front or back over time. (e) Graph showing the relative pump rates (derivative of Ca 2+ change) in the front and the back as a function of local [Ca 2+ ]. The slopes reflect relative pump activity differences in the front and the back for each cell. (f) Statistical analysis of the relative Ca 2+ pump activities in the front versus the back of migrating cells. Bars are mean ± SEM (n = 25 cells). (g) Cells pretreated with inhibitors of PMCA lost their differential Ca 2+ pump activities. Bars are mean ± SEM (n = 47 cells for each group). Student t test was used for Fig. 6a,c,f,g. One-way ANOVA was used for Fig. 6b.
    Figure Legend Snippet: Polarized plasma membrane Ca 2+ pump activity keeps cytosolic Ca 2+ low in the front. (a) Basal cytosolic Ca 2+ levels (measured using Fura-2) in the front were about 50% of the levels in the back of migrating cells (n = 14 cells). Local [Ca 2+ ] was normalized to the average cytosolic level. Cytosolic measurements using GCaMP6s-CAAX are shown in Fig. 1 i,j . (b) High Fura-2 increased the diffusion speed of Ca 2+ , so as to decrease the Ca 2+ gradient between the front and back of migrating cells. Bars are mean ± SEM (n = 160, 160, 80, 160 and 80 cells from left to right for each group). (c) Addition of the PMCA inhibitors Caloxin (200 μM) and La 3+ (200 mM) significantly decreased cytosolic Ca 2+ gradients in migrating cells. Bars are mean ± SEM (n = 47 cells per condition). (d–g) Cytosolic Ca 2+ is transported out of the cell faster in the front than in the back. (d) Addition of thapsigargin, SOC inhibitor BTP2, and Ca 2+ chelator EGTA to migrating HUVECs caused a transient increase in cytosolic Ca 2+ . Local extrusion pump rates were measured as a function of the Ca 2+ level in the front or back over time. (e) Graph showing the relative pump rates (derivative of Ca 2+ change) in the front and the back as a function of local [Ca 2+ ]. The slopes reflect relative pump activity differences in the front and the back for each cell. (f) Statistical analysis of the relative Ca 2+ pump activities in the front versus the back of migrating cells. Bars are mean ± SEM (n = 25 cells). (g) Cells pretreated with inhibitors of PMCA lost their differential Ca 2+ pump activities. Bars are mean ± SEM (n = 47 cells for each group). Student t test was used for Fig. 6a,c,f,g. One-way ANOVA was used for Fig. 6b.

    Techniques Used: Activity Assay, Diffusion-based Assay

    25) Product Images from "Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties"

    Article Title: Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.21.9456-9469.2004

    STC2 is upregulated by the UPR. (A) N2a and PC12 cells were treated with 2 μg of tunicamycin/ml or 300 nM thapsigargin for indicated periods. Total RNA was subject to RT-PCR analyses with STC1 and STC2 primers. UPR was assessed by the induction of BiP mRNA. (B) Quantitative real-time PCR. Steady-state levels of STC2 and β -actin mRNAs were simultaneously quantified in one reaction by using specific primer pairs and fluorescently labeled internal primers. STC2 values were normalized to β -actin mRNA signal and plotted as the fold increase compared to no treatment control. The data represent means ± the SE from three independent experiments. ✽, P
    Figure Legend Snippet: STC2 is upregulated by the UPR. (A) N2a and PC12 cells were treated with 2 μg of tunicamycin/ml or 300 nM thapsigargin for indicated periods. Total RNA was subject to RT-PCR analyses with STC1 and STC2 primers. UPR was assessed by the induction of BiP mRNA. (B) Quantitative real-time PCR. Steady-state levels of STC2 and β -actin mRNAs were simultaneously quantified in one reaction by using specific primer pairs and fluorescently labeled internal primers. STC2 values were normalized to β -actin mRNA signal and plotted as the fold increase compared to no treatment control. The data represent means ± the SE from three independent experiments. ✽, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Labeling

    siRNA inhibition of STC2 induction accentuates cell death by thapsigargin and oxidative stress. (A) N2a cells were cotransfected with empty pSUPER or pSUPER-STC2 siRNA plasmid, along with pEYFP plasmid. Two days after transfection, cells were treated or not treated with 150 nM thapsigargin (Tg), 100 μM H 2 O 2 , 2 μg of tunicamycin/ml (Tm), or 250 nM STS for 16 h. Western blot analysis of STC2, BiP, and β-actin levels. Note the marked reduction in STC2 levels in STC2 siRNA transfected cells. (B) Transfected N2a cells grown on coverslips were washed in PBS and stained with EthD-1 to stain dead cells. Fluorescence images were acquired, and live green cells devoid of EthD-1staining were counted by using MetaMorph software. On the left, the average number of live transfected cells per field in untreated cultures (non) were plotted. On the right, a graph shows the percentage of cell survival (means ± the SE) in cultures treated with various agents (above), normalized to untreated controls in each case, calculated from four independent experiments. ✽, P
    Figure Legend Snippet: siRNA inhibition of STC2 induction accentuates cell death by thapsigargin and oxidative stress. (A) N2a cells were cotransfected with empty pSUPER or pSUPER-STC2 siRNA plasmid, along with pEYFP plasmid. Two days after transfection, cells were treated or not treated with 150 nM thapsigargin (Tg), 100 μM H 2 O 2 , 2 μg of tunicamycin/ml (Tm), or 250 nM STS for 16 h. Western blot analysis of STC2, BiP, and β-actin levels. Note the marked reduction in STC2 levels in STC2 siRNA transfected cells. (B) Transfected N2a cells grown on coverslips were washed in PBS and stained with EthD-1 to stain dead cells. Fluorescence images were acquired, and live green cells devoid of EthD-1staining were counted by using MetaMorph software. On the left, the average number of live transfected cells per field in untreated cultures (non) were plotted. On the right, a graph shows the percentage of cell survival (means ± the SE) in cultures treated with various agents (above), normalized to untreated controls in each case, calculated from four independent experiments. ✽, P

    Techniques Used: Inhibition, Plasmid Preparation, Transfection, Western Blot, Staining, Ethidium Homodimer Assay, Fluorescence, Software

    UPR-mediated expression and secretion of STC2. (A) Specificity of STC2 antiserum. N2a cells were transfected with empty vector or plasmids encoding C-terminally tagged STC1 or untagged STC2. Aliquots of detergent lysates and conditioned media were analyzed by immunoblotting with STC2 antiserum or CT11 (tag) antibody. Note that STC2 antibody does not cross-react with STC1. (B) N2a cells were treated with tunicamycin or thapsigargin for the indicated periods, and detergent lysates were analyzed by blotting with STC2, BiP, CHOP, and β-actin antibodies. Secreted STC2 were immunoprecipitated with STC2 antiserum and then analyzed by immunoblotting with the same antibody. Note the UPR-mediated induction and secretion of STC2.
    Figure Legend Snippet: UPR-mediated expression and secretion of STC2. (A) Specificity of STC2 antiserum. N2a cells were transfected with empty vector or plasmids encoding C-terminally tagged STC1 or untagged STC2. Aliquots of detergent lysates and conditioned media were analyzed by immunoblotting with STC2 antiserum or CT11 (tag) antibody. Note that STC2 antibody does not cross-react with STC1. (B) N2a cells were treated with tunicamycin or thapsigargin for the indicated periods, and detergent lysates were analyzed by blotting with STC2, BiP, CHOP, and β-actin antibodies. Secreted STC2 were immunoprecipitated with STC2 antiserum and then analyzed by immunoblotting with the same antibody. Note the UPR-mediated induction and secretion of STC2.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

    26) Product Images from "Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes"

    Article Title: Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

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

    doi:

    Images showing electroporation of fluo-3 into individual cellular processes of thapsigargin-treated progenitor cells. ( A ) Bright-field image of two progenitor cells. The position of the microelectrodes in relation to the cellular process undergoing permeabilization is shown. ( B ) The same image as A in false-color coded fluorescence (fluorescence intensity proportional to color wavelength) before application of the electric field. ( C – E ) Time sequences starting at 70 s after electroporation (C) and ending 90 s after electroporation (E) . In D and E , the background fluorescence was subtracted from the images. The arrows in C indicate the location of electroporation along the cellular process. In C , right, the process loosened from the substratum after electroporation. The processes were electroporated with three 1-ms pulses of ≈0.5 V.
    Figure Legend Snippet: Images showing electroporation of fluo-3 into individual cellular processes of thapsigargin-treated progenitor cells. ( A ) Bright-field image of two progenitor cells. The position of the microelectrodes in relation to the cellular process undergoing permeabilization is shown. ( B ) The same image as A in false-color coded fluorescence (fluorescence intensity proportional to color wavelength) before application of the electric field. ( C – E ) Time sequences starting at 70 s after electroporation (C) and ending 90 s after electroporation (E) . In D and E , the background fluorescence was subtracted from the images. The arrows in C indicate the location of electroporation along the cellular process. In C , right, the process loosened from the substratum after electroporation. The processes were electroporated with three 1-ms pulses of ≈0.5 V.

    Techniques Used: Electroporation, Fluorescence, Mass Spectrometry

    27) Product Images from "Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca2+ store depletion from store-operated Ca2+ entry"

    Article Title: Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca2+ store depletion from store-operated Ca2+ entry

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200110059

    Pre-activated I SOCE does not inactivate in response to MPF activation. Oocytes were incubated in Ca 2+ -free medium (L-15 with Ca 2+ , buffered at 50 μM), containing thapsigargin (1 μM) to deplete intracellular Ca 2+ store and activate I SOCE . Cells were voltage clamped in a Ca 2+ -free solution (70Mg; see Materials and methods) and then switched to a solution containing 30 mM Ca 2+ (30Ca; see Materials and methods) to induce Ca 2+ influx through SOCE channels. (A) I SOCE and MPF levels from a control thapsigargin–treated oocyte. (B) Oocytes were treated with progesterone (5 μg/ml) for 1 h and then thapsigargin (1 μM) was added, and the cells were incubated until GVBD occurred. I SOCE was measured ∼15 min after GVBD occurrence. I SOCE was still present in this cell, although MPF was activated at high levels, indicating that MPF is unable to block SOCE that has been preactivated before MPF induction. I SOCE (C) and MPF (D) levels in oocytes ( n = 5) and cells treated with progesterone 10–15 min ( n = 8) and 15–30 min after GVBD ( n = 7). I SOCE levels gradually declined after GVBD and MPF levels remained relatively stable.
    Figure Legend Snippet: Pre-activated I SOCE does not inactivate in response to MPF activation. Oocytes were incubated in Ca 2+ -free medium (L-15 with Ca 2+ , buffered at 50 μM), containing thapsigargin (1 μM) to deplete intracellular Ca 2+ store and activate I SOCE . Cells were voltage clamped in a Ca 2+ -free solution (70Mg; see Materials and methods) and then switched to a solution containing 30 mM Ca 2+ (30Ca; see Materials and methods) to induce Ca 2+ influx through SOCE channels. (A) I SOCE and MPF levels from a control thapsigargin–treated oocyte. (B) Oocytes were treated with progesterone (5 μg/ml) for 1 h and then thapsigargin (1 μM) was added, and the cells were incubated until GVBD occurred. I SOCE was measured ∼15 min after GVBD occurrence. I SOCE was still present in this cell, although MPF was activated at high levels, indicating that MPF is unable to block SOCE that has been preactivated before MPF induction. I SOCE (C) and MPF (D) levels in oocytes ( n = 5) and cells treated with progesterone 10–15 min ( n = 8) and 15–30 min after GVBD ( n = 7). I SOCE levels gradually declined after GVBD and MPF levels remained relatively stable.

    Techniques Used: Activation Assay, Incubation, Blocking Assay

    28) Product Images from "CD36 Protein Is Involved in Store-operated Calcium Flux, Phospholipase A2 Activation, and Production of Prostaglandin E2 *"

    Article Title: CD36 Protein Is Involved in Store-operated Calcium Flux, Phospholipase A2 Activation, and Production of Prostaglandin E2 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.232975

    Thapsigargin and UTP induce phosphorylation of ERK1/2 and cPLA 2 α. A , Western analysis profiles. CHO cells stably expressing FLAG-CD36 WT, CD36 K/A, or empty vector ( Vector ) were serum-starved overnight, kept in control medium ( C ), stimulated with
    Figure Legend Snippet: Thapsigargin and UTP induce phosphorylation of ERK1/2 and cPLA 2 α. A , Western analysis profiles. CHO cells stably expressing FLAG-CD36 WT, CD36 K/A, or empty vector ( Vector ) were serum-starved overnight, kept in control medium ( C ), stimulated with

    Techniques Used: Western Blot, Stable Transfection, Expressing, Plasmid Preparation

    CD36 membrane localization and calcium signaling in CHO cells. A , thapsigargin ( TG ) promotes CD36 membrane localization. CHO cells stably expressing FLAG-CD36 wild type ( WT ) or mutated CD36 with alanine substitutions at lysine 469 and lysine 472 ( K/A
    Figure Legend Snippet: CD36 membrane localization and calcium signaling in CHO cells. A , thapsigargin ( TG ) promotes CD36 membrane localization. CHO cells stably expressing FLAG-CD36 wild type ( WT ) or mutated CD36 with alanine substitutions at lysine 469 and lysine 472 ( K/A

    Techniques Used: Stable Transfection, Expressing

    Proposed model for CD36 involvement in calcium influx, cPLA 2 activation, and PGE 2 synthesis. UTP or thapsigargin ( TG ) treatment releases ER calcium via different mechanisms. Thapsigargin inhibits the ER calcium pump SERCA2, and UTP acts via membrane purinergic
    Figure Legend Snippet: Proposed model for CD36 involvement in calcium influx, cPLA 2 activation, and PGE 2 synthesis. UTP or thapsigargin ( TG ) treatment releases ER calcium via different mechanisms. Thapsigargin inhibits the ER calcium pump SERCA2, and UTP acts via membrane purinergic

    Techniques Used: Activation Assay

    Translocation of cPLA 2 α to cellular membranes in response to thapsigargin ( TG ) is dependent on CD36. A , translocation of cPLA 2 α to intracellular membranes. CHO cells expressing either wild-type CD36 or K/A CD36 in control medium ( C ) or
    Figure Legend Snippet: Translocation of cPLA 2 α to cellular membranes in response to thapsigargin ( TG ) is dependent on CD36. A , translocation of cPLA 2 α to intracellular membranes. CHO cells expressing either wild-type CD36 or K/A CD36 in control medium ( C ) or

    Techniques Used: Translocation Assay, Expressing

    29) Product Images from "Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients"

    Article Title: Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-7-11

    Effect of the blockade of progesterone receptor on calcium mobilization evoked by progesterone in human spermatozoa . (A) Fura-2-loaded human spermatozoa from normospermic and asthenozoospermic patients were stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in a calcium-normal solution (1.2 mM [Ca 2+ ] 0 ). (B) Fura-2-loaded human spermatozoa from normospermic patients were pretreated with the anti-progesterone receptor c262 antibody (PR c262) (1:10, final concentration 100 μg/ml for 30 min) or the progesterone receptor antagonist RU-38486 (50 μM for 30 min) and then stimulated with 20 μM progesterone (PROG) in a calcium-normal solution (1.2 mM [Ca 2+ ] 0 ). Traces are representative of 3–4 independent experiments.
    Figure Legend Snippet: Effect of the blockade of progesterone receptor on calcium mobilization evoked by progesterone in human spermatozoa . (A) Fura-2-loaded human spermatozoa from normospermic and asthenozoospermic patients were stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in a calcium-normal solution (1.2 mM [Ca 2+ ] 0 ). (B) Fura-2-loaded human spermatozoa from normospermic patients were pretreated with the anti-progesterone receptor c262 antibody (PR c262) (1:10, final concentration 100 μg/ml for 30 min) or the progesterone receptor antagonist RU-38486 (50 μM for 30 min) and then stimulated with 20 μM progesterone (PROG) in a calcium-normal solution (1.2 mM [Ca 2+ ] 0 ). Traces are representative of 3–4 independent experiments.

    Techniques Used: Concentration Assay

    Effects of cytochalasin D and jasplakinolide on progesterone induced calcium entry in human spermatozoa from asthenozoospermic patients . (A) Fura-2-loaded human spermatozoa were preincubated at room temperature in the presence of 10 μM cytochalasin D (Cyt D) for 40 min or 10 μM jasplakinolide (JP) for 30 min. Cells were then stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in calcium-free medium (+ 100 μM EGTA), and 6 min later CaCl 2 (300 μM) was added to the medium to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, in control, Cyt D-treated and JP-treated spermatozoa, calculated as described in Methods section. Values are means ± SD of seven independent experiments.
    Figure Legend Snippet: Effects of cytochalasin D and jasplakinolide on progesterone induced calcium entry in human spermatozoa from asthenozoospermic patients . (A) Fura-2-loaded human spermatozoa were preincubated at room temperature in the presence of 10 μM cytochalasin D (Cyt D) for 40 min or 10 μM jasplakinolide (JP) for 30 min. Cells were then stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in calcium-free medium (+ 100 μM EGTA), and 6 min later CaCl 2 (300 μM) was added to the medium to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, in control, Cyt D-treated and JP-treated spermatozoa, calculated as described in Methods section. Values are means ± SD of seven independent experiments.

    Techniques Used:

    Mobilization of calcium in response to progesterone in human spermatozoa from normospermic or asthenozoospermic patients . Fura-2-loaded human spermatozoa from normospermic (A) or asthenozoospermic (B) patients were stimulated with 20 μM progesterone alone (PROG) (insets) or plus 1 μM thapsigargin (TG) in calcium-free solution (+ 1 mM EGTA), in the absence (control) or presence of dimethyl BAPTA (10 μM for 30 min). Traces are representative of five independent experiments. (C) Histogram represents the integral for 2.5 min of the calcium release, in normospermic and asthenozoospermic patients, calculated as described in Methods section. Values are means ± SD of five independent experiments. * P
    Figure Legend Snippet: Mobilization of calcium in response to progesterone in human spermatozoa from normospermic or asthenozoospermic patients . Fura-2-loaded human spermatozoa from normospermic (A) or asthenozoospermic (B) patients were stimulated with 20 μM progesterone alone (PROG) (insets) or plus 1 μM thapsigargin (TG) in calcium-free solution (+ 1 mM EGTA), in the absence (control) or presence of dimethyl BAPTA (10 μM for 30 min). Traces are representative of five independent experiments. (C) Histogram represents the integral for 2.5 min of the calcium release, in normospermic and asthenozoospermic patients, calculated as described in Methods section. Values are means ± SD of five independent experiments. * P

    Techniques Used:

    Progesterone induced calcium entry in human spermatozoa from normospermic or asthenozoospermic patients . (A) Fura-2-loaded human spermatozoa were treated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) for 6 min in a calcium-free medium (+ 100 μM EGTA) followed by addition of CaCl 2 (300 μM) to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, in normospermic and asthenozoospermic patients, calculated as described in Methods section. Values are means ± SD of seven independent experiments. * P
    Figure Legend Snippet: Progesterone induced calcium entry in human spermatozoa from normospermic or asthenozoospermic patients . (A) Fura-2-loaded human spermatozoa were treated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) for 6 min in a calcium-free medium (+ 100 μM EGTA) followed by addition of CaCl 2 (300 μM) to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, in normospermic and asthenozoospermic patients, calculated as described in Methods section. Values are means ± SD of seven independent experiments. * P

    Techniques Used:

    Effects of cytochalasin D and jasplakinolide on progesterone induced calcium entry in human spermatozoa from normospermic patients . (A) Fura-2-loaded human spermatozoa were preincubated at room temperature in the presence of 10 μM cytochalasin (Cyt D) for 40 min or 10 μM jasplakinolide (JP) for 30 min. Cells were then stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in calcium-free medium (+ 100 μM EGTA), and 6 min later CaCl 2 (300 μM) was added to the medium to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, calculated as described in Methods section. Values are means ± SD of seven independent experiments. * P
    Figure Legend Snippet: Effects of cytochalasin D and jasplakinolide on progesterone induced calcium entry in human spermatozoa from normospermic patients . (A) Fura-2-loaded human spermatozoa were preincubated at room temperature in the presence of 10 μM cytochalasin (Cyt D) for 40 min or 10 μM jasplakinolide (JP) for 30 min. Cells were then stimulated with 20 μM progesterone (PROG) plus 1 μM thapsigargin (TG) in calcium-free medium (+ 100 μM EGTA), and 6 min later CaCl 2 (300 μM) was added to the medium to initiate calcium entry. Traces are representative of seven independent experiments. (B) Histogram represents the integral for 2.5 min of the amount of calcium entry, calculated as described in Methods section. Values are means ± SD of seven independent experiments. * P

    Techniques Used:

    30) Product Images from "Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation"

    Article Title: Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2017.05.013

    The effect of thapsigargin (TG) on ISO-induced Ca 2+ and pH changes in the ER and mitochondria . ( A , C ) Representative time courses showing the relative intensity changes of ER-pHluorin and ER-R-GECO ( A ) and mito-pHluorin and mito-R-GECO ( C ) following ISO stimulation in BAs pre-treated with 2 μM TG for 30 min. Different phases are marked with shaded areas on the graph. Phase 1 could not be identified due to the lack of a starting point. ( B , D ) Pseudo-color images of cells quantified in A and C , respectively. See Movies S8 (for B ) and S9 (for D ). Scale bars, 10 μm. ( E ) Quantification and comparison of the pH changes in ISO-stimulated BAs without and with TG treatment. Data are means ± SD (mitochondria: n = 14 without TG, n = 5 with TG; ER: n = 10 without TG, n = 6 with TG).
    Figure Legend Snippet: The effect of thapsigargin (TG) on ISO-induced Ca 2+ and pH changes in the ER and mitochondria . ( A , C ) Representative time courses showing the relative intensity changes of ER-pHluorin and ER-R-GECO ( A ) and mito-pHluorin and mito-R-GECO ( C ) following ISO stimulation in BAs pre-treated with 2 μM TG for 30 min. Different phases are marked with shaded areas on the graph. Phase 1 could not be identified due to the lack of a starting point. ( B , D ) Pseudo-color images of cells quantified in A and C , respectively. See Movies S8 (for B ) and S9 (for D ). Scale bars, 10 μm. ( E ) Quantification and comparison of the pH changes in ISO-stimulated BAs without and with TG treatment. Data are means ± SD (mitochondria: n = 14 without TG, n = 5 with TG; ER: n = 10 without TG, n = 6 with TG).

    Techniques Used:

    31) Product Images from "Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during β-adrenergic stimulation in cardiac myocytes"

    Article Title: Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during β-adrenergic stimulation in cardiac myocytes

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvu201

    The effects of thapsigargin on Ca handling and buffering. ( A ) Timecourse. The cell was stimulated with voltage-clamp pulses in the presence of ISO (100 nM). Caffeine/BDM (caff) or thapsigargin (Thap, 5 µM) was applied as indicated. Stimulation was stopped during these periods. ( B ) Measurement of SR Ca content and Ca buffering. The three traces show R / R rest , membrane current, and calculated change of total SR Ca. The left-hand panel was obtained in ISO and the right following 90 s exposure to thapsigargin (5 µM), horizontal bars represent the application of caffeine/BDM. ( C ) Specimen, normalized Ca transients obtained at the times shown by arrows in A . ( D ) SR Ca content in control (left) and thapsigargin (right). ( E ) The bottom graph shows the buffer curves obtained in ISO and thapsigargin with linear regressions. ( F ) Slope of buffer curve in ISO (left) and thapsigargin (right). * P
    Figure Legend Snippet: The effects of thapsigargin on Ca handling and buffering. ( A ) Timecourse. The cell was stimulated with voltage-clamp pulses in the presence of ISO (100 nM). Caffeine/BDM (caff) or thapsigargin (Thap, 5 µM) was applied as indicated. Stimulation was stopped during these periods. ( B ) Measurement of SR Ca content and Ca buffering. The three traces show R / R rest , membrane current, and calculated change of total SR Ca. The left-hand panel was obtained in ISO and the right following 90 s exposure to thapsigargin (5 µM), horizontal bars represent the application of caffeine/BDM. ( C ) Specimen, normalized Ca transients obtained at the times shown by arrows in A . ( D ) SR Ca content in control (left) and thapsigargin (right). ( E ) The bottom graph shows the buffer curves obtained in ISO and thapsigargin with linear regressions. ( F ) Slope of buffer curve in ISO (left) and thapsigargin (right). * P

    Techniques Used:

    32) Product Images from "Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)"

    Article Title: Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)

    Journal: The FASEB Journal

    doi: 10.1096/fj.10-178210

    Effect of SGK1 on SOCE and I CRAC in HEK293 cells expressing Orai1 and STIM1. A ) SOCE in HEK293 cells. Representative tracings of Fura-2 fluorescence ratio in fluorescence spectrometry during and after Ca 2+ depletion and thapsigargin (5 μM) addition
    Figure Legend Snippet: Effect of SGK1 on SOCE and I CRAC in HEK293 cells expressing Orai1 and STIM1. A ) SOCE in HEK293 cells. Representative tracings of Fura-2 fluorescence ratio in fluorescence spectrometry during and after Ca 2+ depletion and thapsigargin (5 μM) addition

    Techniques Used: Expressing, Fluorescence

    33) Product Images from "Proteomics reveals signal peptide features determining the client specificity in human TRAP-dependent ER protein import"

    Article Title: Proteomics reveals signal peptide features determining the client specificity in human TRAP-dependent ER protein import

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06188-z

    TRAP depletion plus live-cell Ca 2+ imaging reveals a TRAP function in Sec61-channel opening. HeLa cells were treated with the indicated siRNAs for 96 h, loaded with Fura 2, and subjected to live-cell imaging of cytosolic Ca 2+ following our established procedure. Ca 2+ release was unmasked by the addition of thapsigargin (TG) in the presence of external EGTA. a , c Average values are presented. Error bars represent standard error of the mean (s.e.m.). b , d Statistical analysis of the changes in cytosolic Ca 2+ after TG addition in a , c . Error bars represent s.e.m. P -values of
    Figure Legend Snippet: TRAP depletion plus live-cell Ca 2+ imaging reveals a TRAP function in Sec61-channel opening. HeLa cells were treated with the indicated siRNAs for 96 h, loaded with Fura 2, and subjected to live-cell imaging of cytosolic Ca 2+ following our established procedure. Ca 2+ release was unmasked by the addition of thapsigargin (TG) in the presence of external EGTA. a , c Average values are presented. Error bars represent standard error of the mean (s.e.m.). b , d Statistical analysis of the changes in cytosolic Ca 2+ after TG addition in a , c . Error bars represent s.e.m. P -values of

    Techniques Used: Imaging, Live Cell Imaging

    34) Product Images from "Transcriptional Control of Human Antigen R by Bone Morphogenetic Protein *"

    Article Title: Transcriptional Control of Human Antigen R by Bone Morphogenetic Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.062216

    Alternate transcriptional starts of HuR mRNA. A , effects of thapsigargin treatment on HuR mRNA expression were assessed by RPA. Lane 1 , molecular size markers; lane 2 , probe + yeast tRNA, undigested; lane 3 , probe + yeast tRNA, digested; lane 4 , mRNA
    Figure Legend Snippet: Alternate transcriptional starts of HuR mRNA. A , effects of thapsigargin treatment on HuR mRNA expression were assessed by RPA. Lane 1 , molecular size markers; lane 2 , probe + yeast tRNA, undigested; lane 3 , probe + yeast tRNA, digested; lane 4 , mRNA

    Techniques Used: Expressing, Recombinase Polymerase Amplification

    35) Product Images from "Stable STIM1 Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation"

    Article Title: Stable STIM1 Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00178

    Knockdown of STIM1 attenuates SOCE in human NPCs. (A,B) Ca 2+ -responses during ER-store release and SOCE induced by Thapsigargin (TG, 10 μM) measured using the ratiometric Ca 2+ -indicator indo-1-AM in wild-type (WT) hNPCs (A) or hNPCs treated with pharmacological inhibitors of SOCE, BTP-2 and 2-APB at the indicated concentrations or DMSO as a solvent control (B) . Each trace represents the mean ±SEM for 25–100 cells. Ionomycin (Iono, 10 μM) was added at the end of each imaging to determine the peak F405/485 ratio obtained after saturation of the Ca 2+ -indicator with Ca 2+ . (C) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated treatment conditions. Mann–Whitney U test with Bonferroni correction. p = 1.819 × 10 -23 for DMSO control compared to BTP-2 treatment and p = 1.442 × 10 -45 for DMSO control compared to 2-APB treatment. (D) (Top) A representative Western blot showing levels of STIM1 protein in hNPCs transduced with an NTC (non-targeting control) or an sh-RNA targeting STIM1 ( STIM1 KD). Actin serves as the loading control. (Bottom) Quantification of STIM1 band intensities normalized to the loading control Actin from three independent biological replicates ( p = 0.00069, Student’s t -test). (E) Ca 2+ -responses during store-release and SOCE in hNPCs transduced with NTC and STIM1 KD. (F) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated genotypes. Peak F405/485 for store-release were not significantly different between NTC and STIM1 KD NPCs. p = 0.0001 for peak F405/485 during SOCE compared between NTC- and STIM1 KD NPCs. (G) Quantification of basal cytosolic [Ca 2+ ] values using Fura-2-AM in NTC- and STIM1 KD NPCs ( p = 1.115 × 10 -8 . Mann–Whitney U test. ( ∗∗∗ Indicates p
    Figure Legend Snippet: Knockdown of STIM1 attenuates SOCE in human NPCs. (A,B) Ca 2+ -responses during ER-store release and SOCE induced by Thapsigargin (TG, 10 μM) measured using the ratiometric Ca 2+ -indicator indo-1-AM in wild-type (WT) hNPCs (A) or hNPCs treated with pharmacological inhibitors of SOCE, BTP-2 and 2-APB at the indicated concentrations or DMSO as a solvent control (B) . Each trace represents the mean ±SEM for 25–100 cells. Ionomycin (Iono, 10 μM) was added at the end of each imaging to determine the peak F405/485 ratio obtained after saturation of the Ca 2+ -indicator with Ca 2+ . (C) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated treatment conditions. Mann–Whitney U test with Bonferroni correction. p = 1.819 × 10 -23 for DMSO control compared to BTP-2 treatment and p = 1.442 × 10 -45 for DMSO control compared to 2-APB treatment. (D) (Top) A representative Western blot showing levels of STIM1 protein in hNPCs transduced with an NTC (non-targeting control) or an sh-RNA targeting STIM1 ( STIM1 KD). Actin serves as the loading control. (Bottom) Quantification of STIM1 band intensities normalized to the loading control Actin from three independent biological replicates ( p = 0.00069, Student’s t -test). (E) Ca 2+ -responses during store-release and SOCE in hNPCs transduced with NTC and STIM1 KD. (F) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated genotypes. Peak F405/485 for store-release were not significantly different between NTC and STIM1 KD NPCs. p = 0.0001 for peak F405/485 during SOCE compared between NTC- and STIM1 KD NPCs. (G) Quantification of basal cytosolic [Ca 2+ ] values using Fura-2-AM in NTC- and STIM1 KD NPCs ( p = 1.115 × 10 -8 . Mann–Whitney U test. ( ∗∗∗ Indicates p

    Techniques Used: Imaging, MANN-WHITNEY, Western Blot, Transduction

    36) Product Images from "Decreased Store Operated Ca2+ Entry in Dendritic Cells Isolated from Mice Expressing PKB/SGK-Resistant GSK3"

    Article Title: Decreased Store Operated Ca2+ Entry in Dendritic Cells Isolated from Mice Expressing PKB/SGK-Resistant GSK3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088637

    Effect of GSK3 inhibitors SB216763 or GSK-XIII on thapsigargin-induced intacellular Ca 2+ release and subsequent SOCE in DCs. A. Representative original tracings showing intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in Fura-2/AM loaded wild type ( gsk3 WT ) dendritic cells (DCs) prior to and following removal of extracellular Ca 2+ , addition of the sarco-endoplasmic Ca 2+ ATPase (SERCA) inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ , all in the absence (open circles) and presence (closed circles) of GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, added 30 min before the experiment) 1 µM (left) or 10 µM (right). B. Representative original tracings showing [Ca 2+ ] i in Fura-2/AM loaded gsk3 WT without (open circles) and with (closed triangles) presence of GSK3 inhibitor GSK-XIII (10 µM, 30 min) DCs prior to and following removal of extracellular Ca 2+ , addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ . C. Arithmetic means ± SEM (n = 16–83) of the peak (left) and slope (right) values of [Ca 2+ ] i increase following addition of thapsigargin reflecting Ca 2+ release from intracellular stores (upper bars) and of [Ca 2+ ] i increase following readdition of extracellular Ca 2+ reflecting store operated Ca 2+ entry (SOCE, lower bars) in gsk3 WT DCs incubated in the presence and absence of GSK3 inhibitor SB216763 (100 nM, 1 µM, 10 µM, 30 min). *(p
    Figure Legend Snippet: Effect of GSK3 inhibitors SB216763 or GSK-XIII on thapsigargin-induced intacellular Ca 2+ release and subsequent SOCE in DCs. A. Representative original tracings showing intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in Fura-2/AM loaded wild type ( gsk3 WT ) dendritic cells (DCs) prior to and following removal of extracellular Ca 2+ , addition of the sarco-endoplasmic Ca 2+ ATPase (SERCA) inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ , all in the absence (open circles) and presence (closed circles) of GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, added 30 min before the experiment) 1 µM (left) or 10 µM (right). B. Representative original tracings showing [Ca 2+ ] i in Fura-2/AM loaded gsk3 WT without (open circles) and with (closed triangles) presence of GSK3 inhibitor GSK-XIII (10 µM, 30 min) DCs prior to and following removal of extracellular Ca 2+ , addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ . C. Arithmetic means ± SEM (n = 16–83) of the peak (left) and slope (right) values of [Ca 2+ ] i increase following addition of thapsigargin reflecting Ca 2+ release from intracellular stores (upper bars) and of [Ca 2+ ] i increase following readdition of extracellular Ca 2+ reflecting store operated Ca 2+ entry (SOCE, lower bars) in gsk3 WT DCs incubated in the presence and absence of GSK3 inhibitor SB216763 (100 nM, 1 µM, 10 µM, 30 min). *(p

    Techniques Used: Incubation

    Thapsigargin-induced intacellular Ca 2+ release and subsequent SOCE in DCs from gsk3 KI and gsk3 WT mice. A. Representative original tracings showing [Ca 2+ ] i in Fura-2/AM loaded gsk3 WT (open circles) and gsk3 KI (closed triangels) DCs prior to and following removal of extracellular Ca 2+ , addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ . B. Arithmetic means ± SEM (n = 44–59) of the peak (left) and slope (right) values of [Ca 2+ ] i increase upon Ca 2+ release from intracellular stores (upper bars) and upon SOCE (lower bars) in gsk3 WT DCs (white bars) and gsk3 KI DCs (black bars). ***(p
    Figure Legend Snippet: Thapsigargin-induced intacellular Ca 2+ release and subsequent SOCE in DCs from gsk3 KI and gsk3 WT mice. A. Representative original tracings showing [Ca 2+ ] i in Fura-2/AM loaded gsk3 WT (open circles) and gsk3 KI (closed triangels) DCs prior to and following removal of extracellular Ca 2+ , addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca 2+ . B. Arithmetic means ± SEM (n = 44–59) of the peak (left) and slope (right) values of [Ca 2+ ] i increase upon Ca 2+ release from intracellular stores (upper bars) and upon SOCE (lower bars) in gsk3 WT DCs (white bars) and gsk3 KI DCs (black bars). ***(p

    Techniques Used: Mouse Assay

    37) Product Images from "INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division"

    Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709111

    Calcium release from the ER triggered by ionomycin and histamine. (A) Time-lapse montages of U2OS cells transfected with the ER calcium probe (ER-GCaMP6-150) and treated with DMSO (top), 4 µM ionomycin (middle), or 100 µM histamine (bottom). Time in seconds. Bar, 10 µm. Corresponds to Video 5. (B) Graph quantifying changes in ER calcium upon the treatments described in A; n = 12, 15, and 15 cells for DMSO, ionomycin, and histamine, respectively. Error bars represent SEM. (C) Effect of thapsigargin on ER calcium (black curves), actin burst (green curves), and mitochondrial calcium (red curves) before and after ionomycin stimulation. U2OS cells transfected with ER calcium probe and mApple-Ftractin. DMSO (top graph) or 1 µM thapsigargin (bottom graph) was applied at 0 s (bold line) and 4 µM ionomycin was applied at the time indicated by the dashed line; n = 10 cells (DMSO) or 13 cells (thapsigargin). Error bars represent SEM. (D) Effect of thapsigargin on histamine-induced cytoplasmic calcium and ER calcium changes. U2OS cells transfected with ER calcium probe and Cyto-R-GECO; n = 15 cells (DMSO) or 15 cells (thapsigargin). (E) Effect of thapsigargin on histamine-induced actin burst and ER calcium. U2OS cells were transfected with ER calcium probe and mApple-Ftractin; n = 10 cells (DMSO) or 11 cells (thapsigargin). Error bars represent SEM. (F) Effect of thapsigargin on histamine-induced mitochondrial calcium and ER calcium. U2OS cells transfected with ER calcium probe and mito-R-GECO; n = 12 cells (DMSO) or 14 cells (Thapsigargin). Error bars represent SEM. (G) Effect of thapsigargin on ionomycin-induced cytoplasmic calcium and ER calcium. U2OS cells transfected with both the Cyto-R-Geco and ER-GCaMP6-150; n = 17 cells (DMSO) or 16 cells (thapsigargin). Error bars represent SEM.
    Figure Legend Snippet: Calcium release from the ER triggered by ionomycin and histamine. (A) Time-lapse montages of U2OS cells transfected with the ER calcium probe (ER-GCaMP6-150) and treated with DMSO (top), 4 µM ionomycin (middle), or 100 µM histamine (bottom). Time in seconds. Bar, 10 µm. Corresponds to Video 5. (B) Graph quantifying changes in ER calcium upon the treatments described in A; n = 12, 15, and 15 cells for DMSO, ionomycin, and histamine, respectively. Error bars represent SEM. (C) Effect of thapsigargin on ER calcium (black curves), actin burst (green curves), and mitochondrial calcium (red curves) before and after ionomycin stimulation. U2OS cells transfected with ER calcium probe and mApple-Ftractin. DMSO (top graph) or 1 µM thapsigargin (bottom graph) was applied at 0 s (bold line) and 4 µM ionomycin was applied at the time indicated by the dashed line; n = 10 cells (DMSO) or 13 cells (thapsigargin). Error bars represent SEM. (D) Effect of thapsigargin on histamine-induced cytoplasmic calcium and ER calcium changes. U2OS cells transfected with ER calcium probe and Cyto-R-GECO; n = 15 cells (DMSO) or 15 cells (thapsigargin). (E) Effect of thapsigargin on histamine-induced actin burst and ER calcium. U2OS cells were transfected with ER calcium probe and mApple-Ftractin; n = 10 cells (DMSO) or 11 cells (thapsigargin). Error bars represent SEM. (F) Effect of thapsigargin on histamine-induced mitochondrial calcium and ER calcium. U2OS cells transfected with ER calcium probe and mito-R-GECO; n = 12 cells (DMSO) or 14 cells (Thapsigargin). Error bars represent SEM. (G) Effect of thapsigargin on ionomycin-induced cytoplasmic calcium and ER calcium. U2OS cells transfected with both the Cyto-R-Geco and ER-GCaMP6-150; n = 17 cells (DMSO) or 16 cells (thapsigargin). Error bars represent SEM.

    Techniques Used: Transfection

    38) Product Images from "Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering"

    Article Title: Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering

    Journal: The EMBO Journal

    doi: 10.15252/embj.201591565

    E-Syt1 plasma membrane binding is triggered by SOCE A–C TIRF microscopy recordings of GCaMP5G (A), mCherry-E-Syt1 (B), and mRFP-STIM1 (C) fluorescence change in HeLa cells in response to release of Ca 2+ from the ER induced by 50 μM BHQ and subsequent activation of SOCE by the addition of 3 mM Ca 2+ . Data are presented as means ± SEM for 55 (GCaMP5G), 39 (E-Syt1), and 39 (STIM1) cells from 4 separate experiments. D Simultaneous TIRF microscopy recordings of GFP-E-Syt1 and mRFP-STIM1 fluorescence from a HeLa cell during release of Ca 2+ from the ER (BHQ) and activation of SOCE (3 Ca 2+ ). The indicated region is shown below on an expanded time scale. E TIRF microscopy images of the HeLa cell in (D). Scale bar is 2 μm. F Immunoblot for STIM1 in control (Ctrl) and STIM1 siRNA-treated HeLa cells. G TIRF microscopy recordings of mCherry-E-Syt1 fluorescence in response to 1 μM thapsigargin (TG) in control (black) and STIM1 siRNA-treated (yellow) HeLa cells (means ± SEM for 31 and 28 cells in 3 separate experiments). H TIRF microscopy recording from a single HeLa cell expressing mCherry-E-Syt1 and the Ca 2+ reporter GCaMP5G and exposed in sequence to the SERCA inhibitor CPA to activate SOCE and then to 1 μM Gd 3+ to stop SOCE without disrupting STIM1-Orai1 contacts. I Quantification of mCh-E-Syt1 PM binding in response to SOCE and the addition of Gd 3+ . Means ± SEM for 24 cells in 3 separate experiments. * P
    Figure Legend Snippet: E-Syt1 plasma membrane binding is triggered by SOCE A–C TIRF microscopy recordings of GCaMP5G (A), mCherry-E-Syt1 (B), and mRFP-STIM1 (C) fluorescence change in HeLa cells in response to release of Ca 2+ from the ER induced by 50 μM BHQ and subsequent activation of SOCE by the addition of 3 mM Ca 2+ . Data are presented as means ± SEM for 55 (GCaMP5G), 39 (E-Syt1), and 39 (STIM1) cells from 4 separate experiments. D Simultaneous TIRF microscopy recordings of GFP-E-Syt1 and mRFP-STIM1 fluorescence from a HeLa cell during release of Ca 2+ from the ER (BHQ) and activation of SOCE (3 Ca 2+ ). The indicated region is shown below on an expanded time scale. E TIRF microscopy images of the HeLa cell in (D). Scale bar is 2 μm. F Immunoblot for STIM1 in control (Ctrl) and STIM1 siRNA-treated HeLa cells. G TIRF microscopy recordings of mCherry-E-Syt1 fluorescence in response to 1 μM thapsigargin (TG) in control (black) and STIM1 siRNA-treated (yellow) HeLa cells (means ± SEM for 31 and 28 cells in 3 separate experiments). H TIRF microscopy recording from a single HeLa cell expressing mCherry-E-Syt1 and the Ca 2+ reporter GCaMP5G and exposed in sequence to the SERCA inhibitor CPA to activate SOCE and then to 1 μM Gd 3+ to stop SOCE without disrupting STIM1-Orai1 contacts. I Quantification of mCh-E-Syt1 PM binding in response to SOCE and the addition of Gd 3+ . Means ± SEM for 24 cells in 3 separate experiments. * P

    Techniques Used: Binding Assay, Microscopy, Fluorescence, Activation Assay, Expressing, Sequencing

    39) Product Images from "Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein *"

    Article Title: Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.535500

    Mobilization of Ca 2+ promotes the degradation of APP CTFs. A and B , cells were incubated in the presence or absence of thapsigargin ( A ) or GPN ( B ) for the indicated periods of time and levels of APP and APP-CTFs analyzed by Western immunoblotting. The
    Figure Legend Snippet: Mobilization of Ca 2+ promotes the degradation of APP CTFs. A and B , cells were incubated in the presence or absence of thapsigargin ( A ) or GPN ( B ) for the indicated periods of time and levels of APP and APP-CTFs analyzed by Western immunoblotting. The

    Techniques Used: Incubation, Western Blot

    40) Product Images from "Endoplasmic reticulum stress in the absence of calnexin"

    Article Title: Endoplasmic reticulum stress in the absence of calnexin

    Journal:

    doi: 10.1007/s12192-008-0049-x

    Induction of UPR in calnexin-deficient cells. a Western blot analysis of chaperone proteins in wild-type and calnexin-deficient cells treated with tunicamycin ( Tn ) and thapsigargin ( Tg ). Duplicate samples were analyzed by Western blotting as indicated
    Figure Legend Snippet: Induction of UPR in calnexin-deficient cells. a Western blot analysis of chaperone proteins in wild-type and calnexin-deficient cells treated with tunicamycin ( Tn ) and thapsigargin ( Tg ). Duplicate samples were analyzed by Western blotting as indicated

    Techniques Used: Western Blot

    Related Articles

    Transfection:

    Article Title: Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells
    Article Snippet: .. Cells grown for 6–12 h on coverslips were incubated for 3 h with the transfection mixture, washed twice with Ca2+ -free PBS, and either cultured in control medium containing 0.1% DMSO or low Ca2+ medium containing 100 nM thapsigargin, or 100 nM thapsigargin plus 5 μM calmidazolium for another 9–11 h. The low Ca2+ medium was prepared as normal growth medium, except that 2 mM EGTA was added to CaCl2 -free DMEM ( GIBCO BRL ). .. The resulting free Ca2+ concentration was calculated to be 60 nM ( ).

    Isolation:

    Article Title: Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response
    Article Snippet: .. Total cellular RNA was isolated from MEFs treated with 1 μM thapsigargin for the indicated number of hours or no stress with the TRIZOL reagent (Invitrogen Life Technologies) in accordance with the manufacturer's instructions. .. A 20-μg portion of RNA from each sample preparation was separated by electrophoresis with a 1.4% agarose-6% formaldehyde gel and visualized by using ethidium bromide staining and UV light.

    Cell Culture:

    Article Title: Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells
    Article Snippet: .. Cells grown for 6–12 h on coverslips were incubated for 3 h with the transfection mixture, washed twice with Ca2+ -free PBS, and either cultured in control medium containing 0.1% DMSO or low Ca2+ medium containing 100 nM thapsigargin, or 100 nM thapsigargin plus 5 μM calmidazolium for another 9–11 h. The low Ca2+ medium was prepared as normal growth medium, except that 2 mM EGTA was added to CaCl2 -free DMEM ( GIBCO BRL ). .. The resulting free Ca2+ concentration was calculated to be 60 nM ( ).

    Incubation:

    Article Title: Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells
    Article Snippet: .. Cells grown for 6–12 h on coverslips were incubated for 3 h with the transfection mixture, washed twice with Ca2+ -free PBS, and either cultured in control medium containing 0.1% DMSO or low Ca2+ medium containing 100 nM thapsigargin, or 100 nM thapsigargin plus 5 μM calmidazolium for another 9–11 h. The low Ca2+ medium was prepared as normal growth medium, except that 2 mM EGTA was added to CaCl2 -free DMEM ( GIBCO BRL ). .. The resulting free Ca2+ concentration was calculated to be 60 nM ( ).

    other:

    Article Title: Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells
    Article Snippet: DiL-C18, thapsigargin and fura-2/AM were from Molecular Probes.

    Article Title: Mechanisms regulating cytochrome c release in pancreatic mitochondria
    Article Snippet: Amplex Red, DHR123, DCF-DA, Fura-2/AM, Fura-6F, MitoTracker Red (CMXRos), TMRM, 1,2-bis( o -aminophenoxy) ethoxy-ethane- N ′-tetraacetic acid (BAPTA-AM), thapsigargin, and Ca2+ /EGTA buffer kits were from Molecular Probes.

    Staining:

    Article Title: CD36 Protein Is Involved in Store-operated Calcium Flux, Phospholipase A2 Activation, and Production of Prostaglandin E2 *
    Article Snippet: .. For CD36 trafficking after thapsigargin treatment, live cells were stained at 4 °C with CD36 antibody for 1 h, washed twice with cold PBS, and then stained at 4 °C with secondary antibody Alexa 594 (Invitrogen) for 40 min. .. The labeled cells were incubated at 37 °C for 10 min with or without 5 μ m thapsigargin, fixed in 3% formaldehyde, and visualized by confocal microscopy.

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    Thermo Fisher gene exp thap1 mm01212603 m1
    <t>Thap1</t> C54Y/+ and Thap1 +/− mice display abnormalities of motor function. ( A ) On the accelerating rotarod testing days, the female Thap1 C54Y/+ performance was significantly worse compared with their Thap1 +/+ littermates (error bars indicate SEM;
    Gene Exp Thap1 Mm01212603 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp thap1 mm01212603 m1/product/Thermo Fisher
    Average 87 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gene exp thap1 mm01212603 m1 - by Bioz Stars, 2020-09
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    99
    Thermo Fisher thapsigargin
    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or <t>thapsigargin</t> (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P
    Thapsigargin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Thermo Fisher
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    Thap1 C54Y/+ and Thap1 +/− mice display abnormalities of motor function. ( A ) On the accelerating rotarod testing days, the female Thap1 C54Y/+ performance was significantly worse compared with their Thap1 +/+ littermates (error bars indicate SEM;

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Thap1 C54Y/+ and Thap1 +/− mice display abnormalities of motor function. ( A ) On the accelerating rotarod testing days, the female Thap1 C54Y/+ performance was significantly worse compared with their Thap1 +/+ littermates (error bars indicate SEM;

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques: Mouse Assay

    Generation of Thap1 C54Y and Thap1 − alleles and mice. ( A ) Schematic representation of THAP1 gene and Thap1 protein with the THAP domain (purple), low-complexity proline-rich region (black), coiled-coil domain (red) and nuclear localization signal

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Generation of Thap1 C54Y and Thap1 − alleles and mice. ( A ) Schematic representation of THAP1 gene and Thap1 protein with the THAP domain (purple), low-complexity proline-rich region (black), coiled-coil domain (red) and nuclear localization signal

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques: Mouse Assay

    Downstream targets of Thap1 are differentially regulated according to genotype and brain region in heterozygote Thap1C54Y/+ and Thap1+/−

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Downstream targets of Thap1 are differentially regulated according to genotype and brain region in heterozygote Thap1C54Y/+ and Thap1+/−

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques:

    Protein levels of torsinA, Gαolf, DARPP-32 and TH are unchanged in Thap1 C54Y/+ and Thap1 + /− mice. ( A ) Western blot analysis of Thap1 in 30 μg of striatal total cellular homogenates showed a significant decrease of the 50-kDa isoform

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Protein levels of torsinA, Gαolf, DARPP-32 and TH are unchanged in Thap1 C54Y/+ and Thap1 + /− mice. ( A ) Western blot analysis of Thap1 in 30 μg of striatal total cellular homogenates showed a significant decrease of the 50-kDa isoform

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques: Mouse Assay, Western Blot

    Thap1 C54Y/+ mice display altered response to injection of the β-adrenergic antagonist propranolol followed by d-amphetamine. ( A ) Locomotor, ambulatory and stereotypy activities of Thap1 C54Y/+ male mice following administration of d-amphetamine

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Thap1 C54Y/+ mice display altered response to injection of the β-adrenergic antagonist propranolol followed by d-amphetamine. ( A ) Locomotor, ambulatory and stereotypy activities of Thap1 C54Y/+ male mice following administration of d-amphetamine

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques: Mouse Assay, Injection

    The projection neurons of the DNC are abnormal in Thap1 C54Y/+ and Thap1 +/− mice. ( A ) Nissl stain of the adult cerebellum (a, a′) shows grossly normal architecture in the adult Thap1 C54Y/+ mouse. Notably, whereas cellularity in the folia

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: The projection neurons of the DNC are abnormal in Thap1 C54Y/+ and Thap1 +/− mice. ( A ) Nissl stain of the adult cerebellum (a, a′) shows grossly normal architecture in the adult Thap1 C54Y/+ mouse. Notably, whereas cellularity in the folia

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques: Mouse Assay, Staining

    Noradrenaline level in the Thap1C54Y/+ and Thap1+/− striatum is increased and response to d-amphetamine plus propranolol is altered in the THAP1C54Y/+ mouse

    Journal: Human Molecular Genetics

    Article Title: Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

    doi: 10.1093/hmg/ddv384

    Figure Lengend Snippet: Noradrenaline level in the Thap1C54Y/+ and Thap1+/− striatum is increased and response to d-amphetamine plus propranolol is altered in the THAP1C54Y/+ mouse

    Article Snippet: RT-qPCR was performed as previously described ( ) using the following Taqman assays: Mm01212603_m1 and Hs00216494_m1 to detect mouse and human Thap1, respectively, Mm00485870_m1 for mouse Rrm1 (ribonucleotide reductase M1), Mm00520052_m1 for Tor1A [torsin family 1, member A (torsin A)], Mm01151063_m1 for TBP (TATA-binding protein), Mm00446968_m1 for HPRT1, Mm02601777_g1 RPS18.

    Techniques:

    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Article Snippet: Apoptosis was induced in primary MEFs by 1 µg/ml tunicamycin, 2 µM thapsigargin, or cultured in the absence of serum or glucose.

    Techniques: Transformation Assay, Infection, Expressing, shRNA, Luciferase, Staining, Transfection

    Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Article Snippet: Apoptosis was induced in primary MEFs by 1 µg/ml tunicamycin, 2 µM thapsigargin, or cultured in the absence of serum or glucose.

    Techniques: Quantitative RT-PCR, Generated, Cell Culture, Staining, Mouse Assay, Irradiation, TUNEL Assay, Light Microscopy, Purification

    Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Article Snippet: Apoptosis was induced in primary MEFs by 1 µg/ml tunicamycin, 2 µM thapsigargin, or cultured in the absence of serum or glucose.

    Techniques: Transformation Assay, Cell Culture, Irradiation, Staining

    PLEK-KO cells reduce TG- or ROS-induced cell death a The image of DIC/DAPI staining. The merged images could be used to determine the proportion of apoptotic cells. b HT-29 cells in the presence of 0.1% DMSO with DAPI staining. b ′ PLEK-KO of control medium. Arrow indicates cell during mitosis (mit) c HT-29 cells in the presence of 1 µM thapsigargin (TG)-treatment for 24 h. Arrowheads indicate fragmented nuclei. c ′ PLEK-KO in 1 µM TG same as c . Less pycnotic cells were observed. d Immunofluorescence using active-caspase-3 antibody/Alexa546 rabbit IgG, FITC-phalloidin, and DAPI. Arrowheads indicate dead cells as measured by the accumulation of active-caspase-3 (depicted in red). d ′ PLEK-KO cells as same as d . e The ratio of cell death after chemical treatments between HT-29 and PLEK-KO cells. The difference of the ratio of apoptosis in TG 24 h and H 2 O 2 24 h were significant (*** P

    Journal: Cell Death Discovery

    Article Title: PLEKHN1 promotes apoptosis by enhancing Bax-Bak hetro-oligomerization through interaction with Bid in human colon cancer

    doi: 10.1038/s41420-017-0006-5

    Figure Lengend Snippet: PLEK-KO cells reduce TG- or ROS-induced cell death a The image of DIC/DAPI staining. The merged images could be used to determine the proportion of apoptotic cells. b HT-29 cells in the presence of 0.1% DMSO with DAPI staining. b ′ PLEK-KO of control medium. Arrow indicates cell during mitosis (mit) c HT-29 cells in the presence of 1 µM thapsigargin (TG)-treatment for 24 h. Arrowheads indicate fragmented nuclei. c ′ PLEK-KO in 1 µM TG same as c . Less pycnotic cells were observed. d Immunofluorescence using active-caspase-3 antibody/Alexa546 rabbit IgG, FITC-phalloidin, and DAPI. Arrowheads indicate dead cells as measured by the accumulation of active-caspase-3 (depicted in red). d ′ PLEK-KO cells as same as d . e The ratio of cell death after chemical treatments between HT-29 and PLEK-KO cells. The difference of the ratio of apoptosis in TG 24 h and H 2 O 2 24 h were significant (*** P

    Article Snippet: Cell lysis and fraction analysis Cells were treated with thapsigargin for 24 h. Prior to cell lysis, the adherent cells were washed with cell rinse buffer (20 mM TrisHCl (pH 7.4), 1.38 mM NaCl) to remove FBS, and treated with or without the bismaleimidohexane (BMH) crosslinker, which has 13 Å spacer, at 5 mM (Thermo Fisher Scientific) for 15 min at room temperature.

    Techniques: Staining, Immunofluorescence

    Cav1.4-deficient T lymphocytes exhibit a reduced Ca 2+ flux PBMCs of patients (n=3) and healthy controls (n=8) were stained with Ca 2+ dyes and different T and B lymphocyte-specific antibodies and analyzed by flow cytometry. Thapsigargin was added to cells after 30 seconds of acquisition. The boxplots show the quantified slopes of increasing Ca 2+ concentration for each cell type (A). The flow cytometry kinetics plots show the actual Ca 2+ influx over time (B). Representative of two technical replicates. * p

    Journal: bioRxiv

    Article Title: Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency

    doi: 10.1101/864280

    Figure Lengend Snippet: Cav1.4-deficient T lymphocytes exhibit a reduced Ca 2+ flux PBMCs of patients (n=3) and healthy controls (n=8) were stained with Ca 2+ dyes and different T and B lymphocyte-specific antibodies and analyzed by flow cytometry. Thapsigargin was added to cells after 30 seconds of acquisition. The boxplots show the quantified slopes of increasing Ca 2+ concentration for each cell type (A). The flow cytometry kinetics plots show the actual Ca 2+ influx over time (B). Representative of two technical replicates. * p

    Article Snippet: 1 μM thapsigargin (Thermo Fisher, cat. nr.

    Techniques: Staining, Flow Cytometry, Concentration Assay