thapsigargin  (Cell Signaling Technology Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Name:
    Thapsigargin
    Description:
    Molecular Weight
    Catalog Number:
    12758
    Price:
    None
    Category:
    Activators Inhibitors
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc thapsigargin
    VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with <t>thapsigargin</t> (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).
    Molecular Weight
    https://www.bioz.com/result/thapsigargin/product/Cell Signaling Technology Inc
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009575

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

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

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

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

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

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

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

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

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

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

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

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

    2) Product Images from "Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response"

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00030-17

    ECD regulates the PERK pathway of the UPR. (A and C) Ecd fl/fl MEFs were infected with an adenovirus coding for GFP (adeno-GFP; control) or Cre (adeno-Cre) for 72 h. The cells were then left untreated or treated with thapsigargin (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) After adenovirus infection as described for panel A, the cells were treated with thapsigargin. Total RNA was isolated and subjected to qRT-PCR with CHOP primers. The data are means and SD for 3 independent experiments. *, P
    Figure Legend Snippet: ECD regulates the PERK pathway of the UPR. (A and C) Ecd fl/fl MEFs were infected with an adenovirus coding for GFP (adeno-GFP; control) or Cre (adeno-Cre) for 72 h. The cells were then left untreated or treated with thapsigargin (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) After adenovirus infection as described for panel A, the cells were treated with thapsigargin. Total RNA was isolated and subjected to qRT-PCR with CHOP primers. The data are means and SD for 3 independent experiments. *, P

    Techniques Used: Infection, SDS Page, Western Blot, Isolation, Quantitative RT-PCR

    Induction of ER stress leads to reduced ECD protein expression in a PERK-eIF2α-dependent manner. (A to C) MCF-10A cells were treated with thapsigargin (Tg; 50 nM) (A) or tunicamycin (Tun; 50 ng/ml) (B), and Panc-1 cells were cultured in a glucose-free medium (C), and then cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved by SDS-PAGE and then subjected to Western blotting with the indicated antibodies. An increase in the level of p-eIF2α served as a marker for induction of ER stress. (D to F) MCF-10A cells were treated with thapsigargin (D and E) and Panc-1 cells were cultured in glucose-free medium (F), and then total RNA was isolated and subjected to qRT-PCR using CHOP primers and ECD primers. Data are means and standard deviations (SD) for 3 independent experiments *, P
    Figure Legend Snippet: Induction of ER stress leads to reduced ECD protein expression in a PERK-eIF2α-dependent manner. (A to C) MCF-10A cells were treated with thapsigargin (Tg; 50 nM) (A) or tunicamycin (Tun; 50 ng/ml) (B), and Panc-1 cells were cultured in a glucose-free medium (C), and then cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved by SDS-PAGE and then subjected to Western blotting with the indicated antibodies. An increase in the level of p-eIF2α served as a marker for induction of ER stress. (D to F) MCF-10A cells were treated with thapsigargin (D and E) and Panc-1 cells were cultured in glucose-free medium (F), and then total RNA was isolated and subjected to qRT-PCR using CHOP primers and ECD primers. Data are means and standard deviations (SD) for 3 independent experiments *, P

    Techniques Used: Expressing, Cell Culture, SDS Page, Western Blot, Marker, Isolation, Quantitative RT-PCR

    Increased induction of GRP78 expression is required for ECD to downregulate PERK signaling. (A) Ecd fl/fl MEFs were treated with adenovirus as described in the legend to Fig. 3A to C , and then the cells were treated with thapsigargin (50 nM). Cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) ECD was knocked down by use of siRNA (20 nM) in Panc-1 cells, followed by exposure to glucose-free medium, and cell lysates were collected at the indicated time points and subjected to Western blotting with the indicated antibodies. (C and D) Following ECD deletion (C) or ECD overexpression (D) and thapsigargin treatment as described above, the levels of GRP78 mRNA were assessed in WT (control) versus ECD −/− (adeno-Cre treated) or control versus ECD-overexpressing MEFs by use of qRT-PCR. (E) ECD was knocked down in Panc-1 cells, followed by cycloheximide treatment (25 μM). Cell lysates were prepared at the indicated time points and subjected to Western blotting with the indicated antibodies. (F) ECD-inducible MEFs and their control MEFs were treated with Dox as described previously, followed by treatment with thapsigargin and then cycloheximide treatment (25 μM) for the indicated times. Cell lysates were prepared and subjected to Western blotting with the indicated antibodies. (G) ECD-overexpressing MEFs and control MEFs were treated with GRP78 siRNA (30 nM) or control siRNA (scrambled). Twenty-four hours later, the cells were treated with Dox for 48 h to induce ECD overexpression, followed by thapsigargin treatment (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and subjected to Western blotting with the indicated antibodies.
    Figure Legend Snippet: Increased induction of GRP78 expression is required for ECD to downregulate PERK signaling. (A) Ecd fl/fl MEFs were treated with adenovirus as described in the legend to Fig. 3A to C , and then the cells were treated with thapsigargin (50 nM). Cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) ECD was knocked down by use of siRNA (20 nM) in Panc-1 cells, followed by exposure to glucose-free medium, and cell lysates were collected at the indicated time points and subjected to Western blotting with the indicated antibodies. (C and D) Following ECD deletion (C) or ECD overexpression (D) and thapsigargin treatment as described above, the levels of GRP78 mRNA were assessed in WT (control) versus ECD −/− (adeno-Cre treated) or control versus ECD-overexpressing MEFs by use of qRT-PCR. (E) ECD was knocked down in Panc-1 cells, followed by cycloheximide treatment (25 μM). Cell lysates were prepared at the indicated time points and subjected to Western blotting with the indicated antibodies. (F) ECD-inducible MEFs and their control MEFs were treated with Dox as described previously, followed by treatment with thapsigargin and then cycloheximide treatment (25 μM) for the indicated times. Cell lysates were prepared and subjected to Western blotting with the indicated antibodies. (G) ECD-overexpressing MEFs and control MEFs were treated with GRP78 siRNA (30 nM) or control siRNA (scrambled). Twenty-four hours later, the cells were treated with Dox for 48 h to induce ECD overexpression, followed by thapsigargin treatment (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and subjected to Western blotting with the indicated antibodies.

    Techniques Used: Expressing, SDS Page, Western Blot, Over Expression, Quantitative RT-PCR

    ECD colocalizes and associates with PERK and GRP78. (A to E) Immortal MEFs were fixed in 3% paraformaldehyde (PFA), stained with the indicated antibodies, and mounted for analyses by structured illumination microscopy (SIM). PERK and GRP78 colocalization served as a positive control. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. (F) MCF-10A cells were fractionated into soluble and microsomal fractions. The purity of the fractions was assessed by using GAPDH (a marker of the soluble/cytoplasmic fraction), PERK and SERCA (both markers of the microsomal fraction), and RCAS1 (a Golgi-predominant protein) as markers ( 88 ). WCL, whole-cell lysate. (G to J) MCF-10A cells were fixed with 3% PFA and stained with the indicated antibodies (all antibodies were generated in rabbits, except for the anti-ECD mouse antibody), followed by species-specific secondary antibodies linked to cDNA probes to allow fluorescent probe-based detection of the PCR amplification products as distinct foci. Incubation with proximity ligation assay plus and minus probes, followed by ligation and amplification, was carried out according to the manufacturer's protocol. Red dots indicate interactions. ECD and PIH1D1 served as positive controls. (K) Lysates of MCF-10A cells, treated with thapsigargin or left untreated, were subjected to IP with anti-ECD antibody (left) or anti-GRP78 (right), followed by Western blotting with the indicated antibodies. ECD and PIH1D1 (left) or GRP78 and PERK (right) served as positive controls.
    Figure Legend Snippet: ECD colocalizes and associates with PERK and GRP78. (A to E) Immortal MEFs were fixed in 3% paraformaldehyde (PFA), stained with the indicated antibodies, and mounted for analyses by structured illumination microscopy (SIM). PERK and GRP78 colocalization served as a positive control. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. (F) MCF-10A cells were fractionated into soluble and microsomal fractions. The purity of the fractions was assessed by using GAPDH (a marker of the soluble/cytoplasmic fraction), PERK and SERCA (both markers of the microsomal fraction), and RCAS1 (a Golgi-predominant protein) as markers ( 88 ). WCL, whole-cell lysate. (G to J) MCF-10A cells were fixed with 3% PFA and stained with the indicated antibodies (all antibodies were generated in rabbits, except for the anti-ECD mouse antibody), followed by species-specific secondary antibodies linked to cDNA probes to allow fluorescent probe-based detection of the PCR amplification products as distinct foci. Incubation with proximity ligation assay plus and minus probes, followed by ligation and amplification, was carried out according to the manufacturer's protocol. Red dots indicate interactions. ECD and PIH1D1 served as positive controls. (K) Lysates of MCF-10A cells, treated with thapsigargin or left untreated, were subjected to IP with anti-ECD antibody (left) or anti-GRP78 (right), followed by Western blotting with the indicated antibodies. ECD and PIH1D1 (left) or GRP78 and PERK (right) served as positive controls.

    Techniques Used: Staining, Microscopy, Positive Control, Marker, Generated, Polymerase Chain Reaction, Amplification, Incubation, Proximity Ligation Assay, Ligation, Western Blot

    ECD overexpression provides a survival advantage. (A) ECD was induced as described in the legends to Fig. 3 and 4 , followed by thapsigargin treatment for 24 h. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) Following ECD induction with Dox and thapsigargin treatment as described above, total RNA was isolated, and CHOP mRNA was assessed by qPCR. The data are means and SD for 3 independent experiments. *, P
    Figure Legend Snippet: ECD overexpression provides a survival advantage. (A) ECD was induced as described in the legends to Fig. 3 and 4 , followed by thapsigargin treatment for 24 h. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) Following ECD induction with Dox and thapsigargin treatment as described above, total RNA was isolated, and CHOP mRNA was assessed by qPCR. The data are means and SD for 3 independent experiments. *, P

    Techniques Used: Over Expression, SDS Page, Western Blot, Isolation, Real-time Polymerase Chain Reaction

    3) Product Images from "Tunicamycin specifically aggravates ER stress and overcomes chemoresistance in multidrug-resistant gastric cancer cells by inhibiting N-glycosylation"

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

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0935-8

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

    Techniques Used: Concentration Assay, Western Blot

    4) Product Images from "Tunicamycin specifically aggravates ER stress and overcomes chemoresistance in multidrug-resistant gastric cancer cells by inhibiting N-glycosylation"

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

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0935-8

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

    Techniques Used: Concentration Assay, Western Blot

    5) Product Images from "Elevated phosphate mediates extensive cellular toxicity: from abnormal proliferation to excessive cell death"

    Article Title: Elevated phosphate mediates extensive cellular toxicity: from abnormal proliferation to excessive cell death

    Journal: bioRxiv

    doi: 10.1101/2020.01.02.892638

    AKT inhibitors aggravates Pi toxicity by inactivating AKT-mTOR signaling, activating ERK signaling and ER stress in HEK293 cells. WB (A) and quantitation (B) analysis shows 5µM MK-2206 and 0.5µM Torin-1 suppressed AKT-mTOR signaling, activated ERK signaling and ER stress in the context of 10mM and 20mM Pi. Thapsigargin (TG) is ER stress enhancer, used as positive control (at concentration of 0.5µM) for ER stress. WB (C) and quantitation (D) analysis shows MK-2206 alleviated Pi suppressed autophagy (reduced ratio of LC-II/LC-I), but had no effect on apoptosis. (E) XTT assay demonstrates MK-2206 abolished 10mM Pi promoted cell proliferation and exacerbated higher Pi (20mM and 40mM) resulted cell loss. Data represent means ± SEM from at least two independent experiments. Unpaired Student t -test was used to compare means between 1mM and higher concentrations of Pi treated groups. Paired t -test was used to compare means between non-treated and MK-2206 treated groups at indicated concentrations of Pi. * P
    Figure Legend Snippet: AKT inhibitors aggravates Pi toxicity by inactivating AKT-mTOR signaling, activating ERK signaling and ER stress in HEK293 cells. WB (A) and quantitation (B) analysis shows 5µM MK-2206 and 0.5µM Torin-1 suppressed AKT-mTOR signaling, activated ERK signaling and ER stress in the context of 10mM and 20mM Pi. Thapsigargin (TG) is ER stress enhancer, used as positive control (at concentration of 0.5µM) for ER stress. WB (C) and quantitation (D) analysis shows MK-2206 alleviated Pi suppressed autophagy (reduced ratio of LC-II/LC-I), but had no effect on apoptosis. (E) XTT assay demonstrates MK-2206 abolished 10mM Pi promoted cell proliferation and exacerbated higher Pi (20mM and 40mM) resulted cell loss. Data represent means ± SEM from at least two independent experiments. Unpaired Student t -test was used to compare means between 1mM and higher concentrations of Pi treated groups. Paired t -test was used to compare means between non-treated and MK-2206 treated groups at indicated concentrations of Pi. * P

    Techniques Used: Western Blot, Quantitation Assay, Positive Control, Concentration Assay, XTT Assay

    6) Product Images from "Mechanisms of lymphoma clearance induced by high-dose alkylating agents"

    Article Title: Mechanisms of lymphoma clearance induced by high-dose alkylating agents

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-18-1393

    Cyclophosphamide Induces ER Stress in DHL cells that Drives Cytokine Secretion (A) Gene-set enrichment analysis (GSEA) of ER-stress signatures from RNA sequencing of DFBL-20954 cells collected from spleens or BM. The comparison reflects genes overexpressed after cyclophosphamide (CTX) treatment as compared to PBS treatment. P-values have a false discovery rate (FDR) adjustment. Abbreviations: ES, enrichment score; NES, normalized enrichment score. (B) Immunoblotting for the indicated targets using lysates from purified, viable BM DFBL-20954 cells collected 16 hours after in vivo treatment with PBS (vehicle), doxorubicin (Dox) or CTX. Each lane represents an individual mouse. (C) Levels of human VEGF-A in cells (left) or media (right) of DFBL-20954 cells isolated from BM and treated ex vivo for 24 hours with DMSO, thapsigargin (TG) or tunicamycin (TM). Two-sided Welch t -test * p
    Figure Legend Snippet: Cyclophosphamide Induces ER Stress in DHL cells that Drives Cytokine Secretion (A) Gene-set enrichment analysis (GSEA) of ER-stress signatures from RNA sequencing of DFBL-20954 cells collected from spleens or BM. The comparison reflects genes overexpressed after cyclophosphamide (CTX) treatment as compared to PBS treatment. P-values have a false discovery rate (FDR) adjustment. Abbreviations: ES, enrichment score; NES, normalized enrichment score. (B) Immunoblotting for the indicated targets using lysates from purified, viable BM DFBL-20954 cells collected 16 hours after in vivo treatment with PBS (vehicle), doxorubicin (Dox) or CTX. Each lane represents an individual mouse. (C) Levels of human VEGF-A in cells (left) or media (right) of DFBL-20954 cells isolated from BM and treated ex vivo for 24 hours with DMSO, thapsigargin (TG) or tunicamycin (TM). Two-sided Welch t -test * p

    Techniques Used: RNA Sequencing Assay, Purification, In Vivo, Isolation, Ex Vivo

    7) Product Images from "NOD1/NOD2 signaling links ER stress with inflammation"

    Article Title: NOD1/NOD2 signaling links ER stress with inflammation

    Journal: Nature

    doi: 10.1038/nature17631

    Only the pro-inflammatory arm of the UPR requires NOD1 and NOD2 ( a and b ) BMDM from Nod1/2 −/− mice and wild type littermates were stimulated with thapsigargin or MDP, and mRNA abundance for Hsp5a ( a ) and Chop ( b ) was quantified (n = 4). ( c ) Expression of SGT1, HSP90 and TRAF2 was detected by Western blot in lysates of thapsigargin-stimulated BMDM from wild-type mice and Nod1/2 −/− mice (n = 3). Detection of tubulin served as a loading control. A representative image for BMDM from one wild-type and one Nod1/2 −/− animal is shown. ( d ) LDH release induced by treatment of BMDM from wild-type mice and Nod1/2 −/− mice (n = 5) with thapsigargin, DTT or KIRA6. ( e ) Stimulation with MDP or DTT induced IL-6 production in BMDM from C57BL/6 mice (wild type) but not in BMDM from Nod1/2 −/− mice (n = 8). ( f ) IL-6 secretion induced by thapsigargin, but not by the canonical NOD2 ligand MDP, was significantly inhibited by ER stress inhibitor TUDCA in BMDM (n = 8). BMDM did not respond to stimulation with a canonical NOD1 ligand (C12-iE-DAP). ( g–k ) BMDM from wild-type mice and Nod1/2 −/− mice (n = 4) were treated with the PERK inhibitor GSK2656157 (GSK) ( g and i ) or the IRE1α RNase inhibitor STF-083010 (STF) ( h and k ) and IL-6 synthesis measured by ELISA ( g and h ) or mRNA analyzed by real-time PCR ( i and j ). Data are presented as mean ± s.e.m.
    Figure Legend Snippet: Only the pro-inflammatory arm of the UPR requires NOD1 and NOD2 ( a and b ) BMDM from Nod1/2 −/− mice and wild type littermates were stimulated with thapsigargin or MDP, and mRNA abundance for Hsp5a ( a ) and Chop ( b ) was quantified (n = 4). ( c ) Expression of SGT1, HSP90 and TRAF2 was detected by Western blot in lysates of thapsigargin-stimulated BMDM from wild-type mice and Nod1/2 −/− mice (n = 3). Detection of tubulin served as a loading control. A representative image for BMDM from one wild-type and one Nod1/2 −/− animal is shown. ( d ) LDH release induced by treatment of BMDM from wild-type mice and Nod1/2 −/− mice (n = 5) with thapsigargin, DTT or KIRA6. ( e ) Stimulation with MDP or DTT induced IL-6 production in BMDM from C57BL/6 mice (wild type) but not in BMDM from Nod1/2 −/− mice (n = 8). ( f ) IL-6 secretion induced by thapsigargin, but not by the canonical NOD2 ligand MDP, was significantly inhibited by ER stress inhibitor TUDCA in BMDM (n = 8). BMDM did not respond to stimulation with a canonical NOD1 ligand (C12-iE-DAP). ( g–k ) BMDM from wild-type mice and Nod1/2 −/− mice (n = 4) were treated with the PERK inhibitor GSK2656157 (GSK) ( g and i ) or the IRE1α RNase inhibitor STF-083010 (STF) ( h and k ) and IL-6 synthesis measured by ELISA ( g and h ) or mRNA analyzed by real-time PCR ( i and j ). Data are presented as mean ± s.e.m.

    Techniques Used: Mouse Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Proinflammatory responses induced by thapsigargin are NOD1/2-dependent ( a–c ) Groups (n = 5) of wild-type mice and Nod1/2 −/− mice were treated with thapsigargin and received either vehicle control of TUDCA. Synthesis of IL-6 ( a ), KC ( b ) and MIP-1β ( c ) in the serum was determined using a Bio-plex cytokine assay. ( d and e ) Wild-type (C57BL/6) mice and Nod1/2 −/− mice (n = 4) were treated with thapsigargin and transcript levels of Il6 determined by quantitative real-time PCR. Data are expressed as fold-increases over vehicle control-treated animals. Data are presented as mean ± s.e.m.
    Figure Legend Snippet: Proinflammatory responses induced by thapsigargin are NOD1/2-dependent ( a–c ) Groups (n = 5) of wild-type mice and Nod1/2 −/− mice were treated with thapsigargin and received either vehicle control of TUDCA. Synthesis of IL-6 ( a ), KC ( b ) and MIP-1β ( c ) in the serum was determined using a Bio-plex cytokine assay. ( d and e ) Wild-type (C57BL/6) mice and Nod1/2 −/− mice (n = 4) were treated with thapsigargin and transcript levels of Il6 determined by quantitative real-time PCR. Data are expressed as fold-increases over vehicle control-treated animals. Data are presented as mean ± s.e.m.

    Techniques Used: Mouse Assay, Cytokine Assay, Real-time Polymerase Chain Reaction

    Thapsigargin induced IL-6 production is dependent on NOD1 and NOD2 ( a–c ) BMDM from Nod1/2 −/− mice and wild type littermates (n = 8) were stimulated with thapsigargin ( a and b ) and/or KIRA6 ( c ) and Il6 mRNA expression ( a ) and IL-6 protein synthesis ( b and c ) were measured. ( d and e ) Nod1/2 −/− mice and wild type littermates (n = 7) were injected with thapsigargin and IL-6 production in the serum ( d ) and Il6 mRNA expression in the spleen ( e ) were determined. Data are presented as mean ± s.e.m.
    Figure Legend Snippet: Thapsigargin induced IL-6 production is dependent on NOD1 and NOD2 ( a–c ) BMDM from Nod1/2 −/− mice and wild type littermates (n = 8) were stimulated with thapsigargin ( a and b ) and/or KIRA6 ( c ) and Il6 mRNA expression ( a ) and IL-6 protein synthesis ( b and c ) were measured. ( d and e ) Nod1/2 −/− mice and wild type littermates (n = 7) were injected with thapsigargin and IL-6 production in the serum ( d ) and Il6 mRNA expression in the spleen ( e ) were determined. Data are presented as mean ± s.e.m.

    Techniques Used: Mouse Assay, Expressing, Injection

    Il6 expression induced by Chlamydia muridarum HeLa cells were stimulated with MDP, thapsigargin or infected with Chlamydia muridarum and treated with KIRA6 or transfected with RIP2DN. Expression of Il6 was determined by quantitative real-time PCR. Data are presented as mean ± s.e.m., (n = 4).
    Figure Legend Snippet: Il6 expression induced by Chlamydia muridarum HeLa cells were stimulated with MDP, thapsigargin or infected with Chlamydia muridarum and treated with KIRA6 or transfected with RIP2DN. Expression of Il6 was determined by quantitative real-time PCR. Data are presented as mean ± s.e.m., (n = 4).

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

    Related Articles

    Protease Inhibitor:

    Article Title: A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models
    Article Snippet: .. U87-MG cells were seeded (~3 × 105 cells/well in 6-well plates) and treated with either vehicle DMSO, control thapsigargin or indicated compound at doses of 5, 10, or 20 μM for 16 h. Whole cell lysates were prepared using RIPA buffer (Cell Signaling) and protease inhibitor cocktail (Cell Signaling). .. Cell lysates were quantified using the Bio-Rad DC Protein Assay.

    Activation Assay:

    Article Title: Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK
    Article Snippet: .. To determine the effect of ER stress activation on LH responsiveness, the GCs were treated with FSK/PMA for 4 h after treatments of 10 μM testosterone, 1 mM metformin and ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 1 mg/ml; Med Chem Express Company, Monmouth Junction, NJ, USA) for 24 h. Meanwhile, the GCs also treated with ER stress inducer thapsigargin (TG, 1 μM; Cell Signaling Technology, Boston, MA, USA) for 1 h before its treatment with FSK/PMA for 4 h. .. COC treatmentsThe COCs were cultured in IVM medium with 10 μM testosterone, 1 mM metformin, 10 μM SB203580, 1 mg/ml TUDCA for 16 h or with 1 μM TG for 1 h. COC expansion areas were measured by ImageJ software (National Institutes of Health, USA) after indicated treatments.

    other:

    Article Title: Tunicamycin specifically aggravates ER stress and overcomes chemoresistance in multidrug-resistant gastric cancer cells by inhibiting N-glycosylation
    Article Snippet: Thapsigargin (Tg, #12758) was from Cell Signaling Technology (Danvers, MA, USA).

    Western Blot:

    Article Title: NOD1/NOD2 signaling links ER stress with inflammation
    Article Snippet: .. Western Blots BMDM stimulated where indicated with 10µM thapsigargin for 24 hours were lysed in lysis buffer (4% SDS, 100mM Tris, 20% glycerol) and 10 µg of protein was analyzed by Western blot using antibodies raised against rabbit TRAF2 (C192, #4724, Cell signaling), rabbit HSP90 (E289, # 4875, Cell signaling), mouse SGT1 (ab60728, Abcam) and rabbit α/β-Tubulin (#2148, Cell signaling). ..

    Lysis:

    Article Title: NOD1/NOD2 signaling links ER stress with inflammation
    Article Snippet: .. Western Blots BMDM stimulated where indicated with 10µM thapsigargin for 24 hours were lysed in lysis buffer (4% SDS, 100mM Tris, 20% glycerol) and 10 µg of protein was analyzed by Western blot using antibodies raised against rabbit TRAF2 (C192, #4724, Cell signaling), rabbit HSP90 (E289, # 4875, Cell signaling), mouse SGT1 (ab60728, Abcam) and rabbit α/β-Tubulin (#2148, Cell signaling). ..

    Chromatin Immunoprecipitation:

    Article Title: The pseudokinase tribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse ? cells
    Article Snippet: .. MIN6 cells under basal condition, treated with 100 nM thapsigargin for 6 hours or 10 μM forskolin for 1 hour, were prepared with the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according the manufacturer’s protocol. .. In each case, the chromatin fractions were incubated with 10 μg of one of the following antibodies at 4°C overnight: anti-CREB (Cell Signaling Technology) or normal Rabbit IgG (provided with SimpleChIP Kit, Cell Signaling Technology).

    Article Title: Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway
    Article Snippet: .. Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation. .. Antibodies against Xbp1 and ATF4 from Santa Cruz Biotechnology (Santa Cruz, CA) as well as a negative control, rabbit IgG, were used for ChIP assay.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Cell Signaling Technology Inc thapsigargin
    VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with <t>thapsigargin</t> (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).
    Thapsigargin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin/product/Cell Signaling Technology Inc
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    thapsigargin - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

    Techniques: Expressing, Real-time Polymerase Chain Reaction

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0009575

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

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

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

    ECD regulates the PERK pathway of the UPR. (A and C) Ecd fl/fl MEFs were infected with an adenovirus coding for GFP (adeno-GFP; control) or Cre (adeno-Cre) for 72 h. The cells were then left untreated or treated with thapsigargin (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) After adenovirus infection as described for panel A, the cells were treated with thapsigargin. Total RNA was isolated and subjected to qRT-PCR with CHOP primers. The data are means and SD for 3 independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    doi: 10.1128/MCB.00030-17

    Figure Lengend Snippet: ECD regulates the PERK pathway of the UPR. (A and C) Ecd fl/fl MEFs were infected with an adenovirus coding for GFP (adeno-GFP; control) or Cre (adeno-Cre) for 72 h. The cells were then left untreated or treated with thapsigargin (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) After adenovirus infection as described for panel A, the cells were treated with thapsigargin. Total RNA was isolated and subjected to qRT-PCR with CHOP primers. The data are means and SD for 3 independent experiments. *, P

    Article Snippet: Thapsigargin and tunicamycin were obtained from Cell Signaling, dissolved in dimethyl sulfoxide (DMSO), and used at the indicated concentrations. siRNA against GRP78 was from Santa Cruz, and cycloheximide was purchased from Sigma.

    Techniques: Infection, SDS Page, Western Blot, Isolation, Quantitative RT-PCR

    Induction of ER stress leads to reduced ECD protein expression in a PERK-eIF2α-dependent manner. (A to C) MCF-10A cells were treated with thapsigargin (Tg; 50 nM) (A) or tunicamycin (Tun; 50 ng/ml) (B), and Panc-1 cells were cultured in a glucose-free medium (C), and then cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved by SDS-PAGE and then subjected to Western blotting with the indicated antibodies. An increase in the level of p-eIF2α served as a marker for induction of ER stress. (D to F) MCF-10A cells were treated with thapsigargin (D and E) and Panc-1 cells were cultured in glucose-free medium (F), and then total RNA was isolated and subjected to qRT-PCR using CHOP primers and ECD primers. Data are means and standard deviations (SD) for 3 independent experiments *, P

    Journal: Molecular and Cellular Biology

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    doi: 10.1128/MCB.00030-17

    Figure Lengend Snippet: Induction of ER stress leads to reduced ECD protein expression in a PERK-eIF2α-dependent manner. (A to C) MCF-10A cells were treated with thapsigargin (Tg; 50 nM) (A) or tunicamycin (Tun; 50 ng/ml) (B), and Panc-1 cells were cultured in a glucose-free medium (C), and then cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved by SDS-PAGE and then subjected to Western blotting with the indicated antibodies. An increase in the level of p-eIF2α served as a marker for induction of ER stress. (D to F) MCF-10A cells were treated with thapsigargin (D and E) and Panc-1 cells were cultured in glucose-free medium (F), and then total RNA was isolated and subjected to qRT-PCR using CHOP primers and ECD primers. Data are means and standard deviations (SD) for 3 independent experiments *, P

    Article Snippet: Thapsigargin and tunicamycin were obtained from Cell Signaling, dissolved in dimethyl sulfoxide (DMSO), and used at the indicated concentrations. siRNA against GRP78 was from Santa Cruz, and cycloheximide was purchased from Sigma.

    Techniques: Expressing, Cell Culture, SDS Page, Western Blot, Marker, Isolation, Quantitative RT-PCR

    Increased induction of GRP78 expression is required for ECD to downregulate PERK signaling. (A) Ecd fl/fl MEFs were treated with adenovirus as described in the legend to Fig. 3A to C , and then the cells were treated with thapsigargin (50 nM). Cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) ECD was knocked down by use of siRNA (20 nM) in Panc-1 cells, followed by exposure to glucose-free medium, and cell lysates were collected at the indicated time points and subjected to Western blotting with the indicated antibodies. (C and D) Following ECD deletion (C) or ECD overexpression (D) and thapsigargin treatment as described above, the levels of GRP78 mRNA were assessed in WT (control) versus ECD −/− (adeno-Cre treated) or control versus ECD-overexpressing MEFs by use of qRT-PCR. (E) ECD was knocked down in Panc-1 cells, followed by cycloheximide treatment (25 μM). Cell lysates were prepared at the indicated time points and subjected to Western blotting with the indicated antibodies. (F) ECD-inducible MEFs and their control MEFs were treated with Dox as described previously, followed by treatment with thapsigargin and then cycloheximide treatment (25 μM) for the indicated times. Cell lysates were prepared and subjected to Western blotting with the indicated antibodies. (G) ECD-overexpressing MEFs and control MEFs were treated with GRP78 siRNA (30 nM) or control siRNA (scrambled). Twenty-four hours later, the cells were treated with Dox for 48 h to induce ECD overexpression, followed by thapsigargin treatment (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and subjected to Western blotting with the indicated antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    doi: 10.1128/MCB.00030-17

    Figure Lengend Snippet: Increased induction of GRP78 expression is required for ECD to downregulate PERK signaling. (A) Ecd fl/fl MEFs were treated with adenovirus as described in the legend to Fig. 3A to C , and then the cells were treated with thapsigargin (50 nM). Cell lysates were prepared at the indicated time points. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) ECD was knocked down by use of siRNA (20 nM) in Panc-1 cells, followed by exposure to glucose-free medium, and cell lysates were collected at the indicated time points and subjected to Western blotting with the indicated antibodies. (C and D) Following ECD deletion (C) or ECD overexpression (D) and thapsigargin treatment as described above, the levels of GRP78 mRNA were assessed in WT (control) versus ECD −/− (adeno-Cre treated) or control versus ECD-overexpressing MEFs by use of qRT-PCR. (E) ECD was knocked down in Panc-1 cells, followed by cycloheximide treatment (25 μM). Cell lysates were prepared at the indicated time points and subjected to Western blotting with the indicated antibodies. (F) ECD-inducible MEFs and their control MEFs were treated with Dox as described previously, followed by treatment with thapsigargin and then cycloheximide treatment (25 μM) for the indicated times. Cell lysates were prepared and subjected to Western blotting with the indicated antibodies. (G) ECD-overexpressing MEFs and control MEFs were treated with GRP78 siRNA (30 nM) or control siRNA (scrambled). Twenty-four hours later, the cells were treated with Dox for 48 h to induce ECD overexpression, followed by thapsigargin treatment (50 nM). Equal amounts of proteins were resolved in an SDS-PAGE gel and subjected to Western blotting with the indicated antibodies.

    Article Snippet: Thapsigargin and tunicamycin were obtained from Cell Signaling, dissolved in dimethyl sulfoxide (DMSO), and used at the indicated concentrations. siRNA against GRP78 was from Santa Cruz, and cycloheximide was purchased from Sigma.

    Techniques: Expressing, SDS Page, Western Blot, Over Expression, Quantitative RT-PCR

    ECD colocalizes and associates with PERK and GRP78. (A to E) Immortal MEFs were fixed in 3% paraformaldehyde (PFA), stained with the indicated antibodies, and mounted for analyses by structured illumination microscopy (SIM). PERK and GRP78 colocalization served as a positive control. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. (F) MCF-10A cells were fractionated into soluble and microsomal fractions. The purity of the fractions was assessed by using GAPDH (a marker of the soluble/cytoplasmic fraction), PERK and SERCA (both markers of the microsomal fraction), and RCAS1 (a Golgi-predominant protein) as markers ( 88 ). WCL, whole-cell lysate. (G to J) MCF-10A cells were fixed with 3% PFA and stained with the indicated antibodies (all antibodies were generated in rabbits, except for the anti-ECD mouse antibody), followed by species-specific secondary antibodies linked to cDNA probes to allow fluorescent probe-based detection of the PCR amplification products as distinct foci. Incubation with proximity ligation assay plus and minus probes, followed by ligation and amplification, was carried out according to the manufacturer's protocol. Red dots indicate interactions. ECD and PIH1D1 served as positive controls. (K) Lysates of MCF-10A cells, treated with thapsigargin or left untreated, were subjected to IP with anti-ECD antibody (left) or anti-GRP78 (right), followed by Western blotting with the indicated antibodies. ECD and PIH1D1 (left) or GRP78 and PERK (right) served as positive controls.

    Journal: Molecular and Cellular Biology

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    doi: 10.1128/MCB.00030-17

    Figure Lengend Snippet: ECD colocalizes and associates with PERK and GRP78. (A to E) Immortal MEFs were fixed in 3% paraformaldehyde (PFA), stained with the indicated antibodies, and mounted for analyses by structured illumination microscopy (SIM). PERK and GRP78 colocalization served as a positive control. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. (F) MCF-10A cells were fractionated into soluble and microsomal fractions. The purity of the fractions was assessed by using GAPDH (a marker of the soluble/cytoplasmic fraction), PERK and SERCA (both markers of the microsomal fraction), and RCAS1 (a Golgi-predominant protein) as markers ( 88 ). WCL, whole-cell lysate. (G to J) MCF-10A cells were fixed with 3% PFA and stained with the indicated antibodies (all antibodies were generated in rabbits, except for the anti-ECD mouse antibody), followed by species-specific secondary antibodies linked to cDNA probes to allow fluorescent probe-based detection of the PCR amplification products as distinct foci. Incubation with proximity ligation assay plus and minus probes, followed by ligation and amplification, was carried out according to the manufacturer's protocol. Red dots indicate interactions. ECD and PIH1D1 served as positive controls. (K) Lysates of MCF-10A cells, treated with thapsigargin or left untreated, were subjected to IP with anti-ECD antibody (left) or anti-GRP78 (right), followed by Western blotting with the indicated antibodies. ECD and PIH1D1 (left) or GRP78 and PERK (right) served as positive controls.

    Article Snippet: Thapsigargin and tunicamycin were obtained from Cell Signaling, dissolved in dimethyl sulfoxide (DMSO), and used at the indicated concentrations. siRNA against GRP78 was from Santa Cruz, and cycloheximide was purchased from Sigma.

    Techniques: Staining, Microscopy, Positive Control, Marker, Generated, Polymerase Chain Reaction, Amplification, Incubation, Proximity Ligation Assay, Ligation, Western Blot

    ECD overexpression provides a survival advantage. (A) ECD was induced as described in the legends to Fig. 3 and 4 , followed by thapsigargin treatment for 24 h. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) Following ECD induction with Dox and thapsigargin treatment as described above, total RNA was isolated, and CHOP mRNA was assessed by qPCR. The data are means and SD for 3 independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

    doi: 10.1128/MCB.00030-17

    Figure Lengend Snippet: ECD overexpression provides a survival advantage. (A) ECD was induced as described in the legends to Fig. 3 and 4 , followed by thapsigargin treatment for 24 h. Equal amounts of proteins were resolved in an SDS-PAGE gel and then subjected to Western blotting with the indicated antibodies. (B) Following ECD induction with Dox and thapsigargin treatment as described above, total RNA was isolated, and CHOP mRNA was assessed by qPCR. The data are means and SD for 3 independent experiments. *, P

    Article Snippet: Thapsigargin and tunicamycin were obtained from Cell Signaling, dissolved in dimethyl sulfoxide (DMSO), and used at the indicated concentrations. siRNA against GRP78 was from Santa Cruz, and cycloheximide was purchased from Sigma.

    Techniques: Over Expression, SDS Page, Western Blot, Isolation, Real-time Polymerase Chain Reaction

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

    Journal: Journal of Experimental & Clinical Cancer Research : CR

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

    doi: 10.1186/s13046-018-0935-8

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

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

    Techniques: Concentration Assay, Western Blot

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

    Journal: Journal of Experimental & Clinical Cancer Research : CR

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

    doi: 10.1186/s13046-018-0935-8

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

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

    Techniques: Concentration Assay, Western Blot