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  • 97
    Thermo Fisher thapsigargin
    ATF3 facilitates enhanced GADD34 expression in response to ER stress and amino acid starvation. ATF3 +/+ and ATF3 −/− MEF cells were treated with <t>thapsigargin</t> (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.
    Thapsigargin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore thapsigargin thap
    Inhibition of mitochondrial Hsp90s sensitizes HeLa cells toward <t>thapsigargin.</t> (A) Cytoplasmic calcium and mitochondrial membrane potential by suboptimal dose of gamitrinib. Fluo-4 or TMRM/MitoTracker-labeled HeLa cells were incubated with 5 μM gamitrinib for 24 hours and analyzed by confocal microscope. Bar, 20 μm. (B) Combination effect in HeLa. HeLa cells were treated with various concentrations of Thap in the presence of 5 μM of either 17AAG or gamitrinib, and analyzed by MTT assay (left). Alternatively, HeLa cells were treated with 5 μM gamitrinib and/or 0.06 μM Thap for 24 hours and analyzed by the MTT assay. ***, p
    Thapsigargin Thap, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam thap sigargin thap
    Dexamethasone stimulates intracellular Ca2+ release and SOCE and co-treatment with dexamethasone and SOC inhibitors markedly enhances ALL cells death Cells were loaded with Fura-2/AM, and the changes in intracellular Ca 2+ , [Ca 2+ ]i, (F340/F380) were monitored. (A, B) The colored time-lapse images and the graphical representation show the changes in [Ca 2+ ]i evoked by dexamethasone (Dex), in Reh and Nalm-6 cell lines, respectively, in Ca 2+ -containing and in Ca 2+ -free buffer. (C, D) ALL cells before exposure to 100 nM dexamethasone (Dex) in Ca 2+ buffer were preincubated (20 minutes) with U73122 (10 μM), SKF96365 (10 μM) or 2-APB (10 μM). Data are mean ± SEM (n = 5). (E) Effect of dexamethasone (Dex) on SOCE activation in ALL cells. ALL cells ER calcium stores were depleted with <t>thapsigargin</t> (TG, 1 μM) in calcium-free suspension medium in the presence or absence of 2-APB (10 μM), cells were then treated without or with 100 nM dexamethasone, followed by addition of 1.8 mM CaCl 2 . Data are mean ± SEM (n = 3). Reh (F) and Nalm-6 (G) cells were treated with SOC inhibitors (SKF 96365, 5 μM and 2-APB, 5 μM) and dexamethasone (100 nM) alone or in combination for 48 h. Cell death was detected by MTT metabolic colorimetric assay. Data are representative of triplicate experiments. *p
    Thap Sigargin Thap, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Adipogen thapsigargin thap
    Dexamethasone stimulates intracellular Ca2+ release and SOCE and co-treatment with dexamethasone and SOC inhibitors markedly enhances ALL cells death Cells were loaded with Fura-2/AM, and the changes in intracellular Ca 2+ , [Ca 2+ ]i, (F340/F380) were monitored. (A, B) The colored time-lapse images and the graphical representation show the changes in [Ca 2+ ]i evoked by dexamethasone (Dex), in Reh and Nalm-6 cell lines, respectively, in Ca 2+ -containing and in Ca 2+ -free buffer. (C, D) ALL cells before exposure to 100 nM dexamethasone (Dex) in Ca 2+ buffer were preincubated (20 minutes) with U73122 (10 μM), SKF96365 (10 μM) or 2-APB (10 μM). Data are mean ± SEM (n = 5). (E) Effect of dexamethasone (Dex) on SOCE activation in ALL cells. ALL cells ER calcium stores were depleted with <t>thapsigargin</t> (TG, 1 μM) in calcium-free suspension medium in the presence or absence of 2-APB (10 μM), cells were then treated without or with 100 nM dexamethasone, followed by addition of 1.8 mM CaCl 2 . Data are mean ± SEM (n = 3). Reh (F) and Nalm-6 (G) cells were treated with SOC inhibitors (SKF 96365, 5 μM and 2-APB, 5 μM) and dexamethasone (100 nM) alone or in combination for 48 h. Cell death was detected by MTT metabolic colorimetric assay. Data are representative of triplicate experiments. *p
    Thapsigargin Thap, supplied by Adipogen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Applichem thapsigargin thap
    Dexamethasone stimulates intracellular Ca2+ release and SOCE and co-treatment with dexamethasone and SOC inhibitors markedly enhances ALL cells death Cells were loaded with Fura-2/AM, and the changes in intracellular Ca 2+ , [Ca 2+ ]i, (F340/F380) were monitored. (A, B) The colored time-lapse images and the graphical representation show the changes in [Ca 2+ ]i evoked by dexamethasone (Dex), in Reh and Nalm-6 cell lines, respectively, in Ca 2+ -containing and in Ca 2+ -free buffer. (C, D) ALL cells before exposure to 100 nM dexamethasone (Dex) in Ca 2+ buffer were preincubated (20 minutes) with U73122 (10 μM), SKF96365 (10 μM) or 2-APB (10 μM). Data are mean ± SEM (n = 5). (E) Effect of dexamethasone (Dex) on SOCE activation in ALL cells. ALL cells ER calcium stores were depleted with <t>thapsigargin</t> (TG, 1 μM) in calcium-free suspension medium in the presence or absence of 2-APB (10 μM), cells were then treated without or with 100 nM dexamethasone, followed by addition of 1.8 mM CaCl 2 . Data are mean ± SEM (n = 3). Reh (F) and Nalm-6 (G) cells were treated with SOC inhibitors (SKF 96365, 5 μM and 2-APB, 5 μM) and dexamethasone (100 nM) alone or in combination for 48 h. Cell death was detected by MTT metabolic colorimetric assay. Data are representative of triplicate experiments. *p
    Thapsigargin Thap, supplied by Applichem, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc thapsigargin
    VEGFA mRNA expression is induced by ER stress. Expression levels of VEGFA, Spliced Xbp1 and BiP were measured by quantitative PCR in PC3 cells, HepG2 cells and INS-1 832/13 cells following treatment with <t>thapsigargin</t> (Tg, 1 µM), tunicamycin (Tm, 5 µg/ml) and untreated (UT) control for 4 hr (n = 3, values are mean ± SD).
    Thapsigargin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Tocris thapsigargin
    GABA B -receptor activationmay operate two distinct pathways to activate or inhibit the iLTP induction. (A) Left, schematic representation of the working model of CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in wt Purkinje cell. The model is proposed based on previous studies ( Kano et al., 1996 ; Kawaguchi and Hirano, 2002 ) and the current data. Cascades are simplified for the clarity of illustration. Arrows indicate activation cascades, bars indicate inhibitory cascades. Note that in the presence of both α and βCaMKII, the calcium release from internal stores upon GABA B -receptor activation is outcompeted by the suppressing PKA-PP1 pathway (dashed arrow). AC, adenylyl cyclase; D32, DARPP-32. Right, schematic representation of the CaMKII mediated iLTP induction cascade and GABA B -receptor mediated inhibition of iLTP in Camk2b - / - Purkinje cells. Genetic deletion of βCaMKII revealed a rescue of iLTP by GABA B -receptor activation. Note that (1) the inhibitory effect of PKA-PP1 pathway upon GABA B -receptor activation is minimized (indicated in dashed lines) in the absence βCaMKII and that (2) the facilitating effects of calcium release from internal stores enables the rescue of iLTP. (B) Inhibition of PKA with KT5720 suppresses iLTP in wt Purkinje cells ( n = 5), but does not rescue iLTP in Camk2b - / - Purkinje cells ( n = 6) following CF stimulation. (C) Inhibition of calcium release from internal stores with <t>thapsigargin</t> abolishes the facilitation of iLTP in Camk2b - / - Purkinje cells ( n = 7) following paired MLI-CF stimulation, as well as iLTP in wt Purkinje cells ( n = 6) following CF stimulation. Error bars represent SEM. Asterisks with brackets indicate statistical significance between wt and knockout mice.
    Thapsigargin, supplied by Tocris, used in various techniques. Bioz Stars score: 97/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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).

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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).

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.3.1365-1377.2004

    Figure Lengend 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.

    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.

    Techniques: Expressing, Transfection, Plasmid Preparation

    Integrated peak area coefficient of variation values across: ( a ) all peptide transitions (n = 305) for each bacterial dilution and ( b ) 10 transitions with the lowest sum of CV across all samples, representing the most reliable transitions (Supplementary Table S5 ). These 10 peptide transitions with the lowest CV correspond to 7 peptides from 7 proteins involved in diverse functions (e.g., purine metabolism, fatty acid biosynthesis, transcription). X-axis values correspond to the bacterial dilutions, indicated by cellular ratio Rpom:1 Thaps cell. The boxes represent the upper and lower quartiles of the data distribution; the horizontal black line represents median value; “whiskers” extend to the greatest and least values, excluding outliers; open circles represent outliers (±1.5 times the upper or lower quartiles).

    Journal: Scientific Reports

    Article Title: MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides

    doi: 10.1038/s41598-018-27650-4

    Figure Lengend Snippet: Integrated peak area coefficient of variation values across: ( a ) all peptide transitions (n = 305) for each bacterial dilution and ( b ) 10 transitions with the lowest sum of CV across all samples, representing the most reliable transitions (Supplementary Table S5 ). These 10 peptide transitions with the lowest CV correspond to 7 peptides from 7 proteins involved in diverse functions (e.g., purine metabolism, fatty acid biosynthesis, transcription). X-axis values correspond to the bacterial dilutions, indicated by cellular ratio Rpom:1 Thaps cell. The boxes represent the upper and lower quartiles of the data distribution; the horizontal black line represents median value; “whiskers” extend to the greatest and least values, excluding outliers; open circles represent outliers (±1.5 times the upper or lower quartiles).

    Article Snippet: The three technical replicates from the DDA experiment for the dilution of 5000 Rpom: 1 Thaps cell were pooled in equal quantities to create two new technical replicates for data independent acquisition (DIA) on the QE (Thermo).

    Techniques:

    Illustration of experimental setup and workflow for mass spectrometry data acquisition and analysis. ( a ) Serial dilutions were completed using bacterial cells (RPom) as the diluent (see text). Dilution was based on cell counts to achieve cellular rations of Rpom ( R . pomeroyi ) to Thaps ( T . pseudonana ). Each serial dilution was then lysed and proteins were digested prior to MS experiments. ( b ) MS experimental workflow: 1. Data dependent acquisition (DDA) was performed on the Q-Exactive-HF (QE) to assess the limit of detection for a standard, discovery-driven proteomics experiment. 2. Data independent acquisition (DIA) was also completed on the QE to create spectral libraries for selected reaction monitoring (SRM) method development. 3 4. These spectral libraries were analyzed with PECAN and Skyline was used to select optimal transitions and to design an instrument method for SRM analyses. 5. SRM was completed on the TSQ Vantage for 309 bacterial peptide transitions. 6. Peptide transition detection and quantification was performed in Skyline. ( c ) The chromatograms of peptide IPSAVGYQPTLATDMGAMQER (from protein Q5LNP1) were collected using the 3 different MS approaches (DDA, DIA, and SRM) on bacterial dilution 5000:1. Black vertical lines indicate peak integration boundaries, and colored peaks represent the different transitions (i.e. peptide fragments) collected.

    Journal: Scientific Reports

    Article Title: MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides

    doi: 10.1038/s41598-018-27650-4

    Figure Lengend Snippet: Illustration of experimental setup and workflow for mass spectrometry data acquisition and analysis. ( a ) Serial dilutions were completed using bacterial cells (RPom) as the diluent (see text). Dilution was based on cell counts to achieve cellular rations of Rpom ( R . pomeroyi ) to Thaps ( T . pseudonana ). Each serial dilution was then lysed and proteins were digested prior to MS experiments. ( b ) MS experimental workflow: 1. Data dependent acquisition (DDA) was performed on the Q-Exactive-HF (QE) to assess the limit of detection for a standard, discovery-driven proteomics experiment. 2. Data independent acquisition (DIA) was also completed on the QE to create spectral libraries for selected reaction monitoring (SRM) method development. 3 4. These spectral libraries were analyzed with PECAN and Skyline was used to select optimal transitions and to design an instrument method for SRM analyses. 5. SRM was completed on the TSQ Vantage for 309 bacterial peptide transitions. 6. Peptide transition detection and quantification was performed in Skyline. ( c ) The chromatograms of peptide IPSAVGYQPTLATDMGAMQER (from protein Q5LNP1) were collected using the 3 different MS approaches (DDA, DIA, and SRM) on bacterial dilution 5000:1. Black vertical lines indicate peak integration boundaries, and colored peaks represent the different transitions (i.e. peptide fragments) collected.

    Article Snippet: The three technical replicates from the DDA experiment for the dilution of 5000 Rpom: 1 Thaps cell were pooled in equal quantities to create two new technical replicates for data independent acquisition (DIA) on the QE (Thermo).

    Techniques: Mass Spectrometry, Serial Dilution

    Peptide spectral matches (PSMs) from data dependent acquisition (DDA) of bacterial dilutions in phytoplankton. Points are colored by the cellular ratio of Rpom:1 Thaps cell, indicated in the legends. Number of Thaps peptide spectral matches (PSMs) per injection is plotted against number of Rpom PSMs. The insert shows a detailed view of the upper left-hand corner of the graph.

    Journal: Scientific Reports

    Article Title: MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides

    doi: 10.1038/s41598-018-27650-4

    Figure Lengend Snippet: Peptide spectral matches (PSMs) from data dependent acquisition (DDA) of bacterial dilutions in phytoplankton. Points are colored by the cellular ratio of Rpom:1 Thaps cell, indicated in the legends. Number of Thaps peptide spectral matches (PSMs) per injection is plotted against number of Rpom PSMs. The insert shows a detailed view of the upper left-hand corner of the graph.

    Article Snippet: The three technical replicates from the DDA experiment for the dilution of 5000 Rpom: 1 Thaps cell were pooled in equal quantities to create two new technical replicates for data independent acquisition (DIA) on the QE (Thermo).

    Techniques: Injection

    Nonmetric multidimensional scaling plots (NMDS) of proteomic data (normalized spectral abundance factor) from Rpom:Thaps dilutions for DDA ( a ) and SRM ( b ) analyses. Points are colored by cellular ratio Rpom:1 Thaps cell.

    Journal: Scientific Reports

    Article Title: MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides

    doi: 10.1038/s41598-018-27650-4

    Figure Lengend Snippet: Nonmetric multidimensional scaling plots (NMDS) of proteomic data (normalized spectral abundance factor) from Rpom:Thaps dilutions for DDA ( a ) and SRM ( b ) analyses. Points are colored by cellular ratio Rpom:1 Thaps cell.

    Article Snippet: The three technical replicates from the DDA experiment for the dilution of 5000 Rpom: 1 Thaps cell were pooled in equal quantities to create two new technical replicates for data independent acquisition (DIA) on the QE (Thermo).

    Techniques:

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

    Journal: Molecular Cancer

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

    doi: 10.1186/1476-4598-13-148

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

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

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

    Hcy upregulates Ero1α and induces ER stress and H 2 O 2 accumulation in HUVECs. (A) HUVECs were treated without or with different concentrations of Hcy or 5 μM Thaps for 12 h. Cell lysates were subjected to immunoblotting and statistical analysis for Ero1α and BiP expression. Representative blots were shown. (B) HUVECs were treated with 200 μM Hcy for a different time as indicated or with 5 μM Thaps for 8 h. Cell lysates were subjected to immunoblotting and statistical analysis for expression of Ero1α, BiP, XBP1s/XBP1u, p-eIF2α/eIF2α, and CHOP. Representative blots were shown. (A-B) Data were shown as mean ± SEM from three independent experiments, *p

    Journal: Redox Biology

    Article Title: Homocysteine causes vascular endothelial dysfunction by disrupting endoplasmic reticulum redox homeostasis

    doi: 10.1016/j.redox.2018.09.021

    Figure Lengend Snippet: Hcy upregulates Ero1α and induces ER stress and H 2 O 2 accumulation in HUVECs. (A) HUVECs were treated without or with different concentrations of Hcy or 5 μM Thaps for 12 h. Cell lysates were subjected to immunoblotting and statistical analysis for Ero1α and BiP expression. Representative blots were shown. (B) HUVECs were treated with 200 μM Hcy for a different time as indicated or with 5 μM Thaps for 8 h. Cell lysates were subjected to immunoblotting and statistical analysis for expression of Ero1α, BiP, XBP1s/XBP1u, p-eIF2α/eIF2α, and CHOP. Representative blots were shown. (A-B) Data were shown as mean ± SEM from three independent experiments, *p

    Article Snippet: 4.1 Reagents and antibodies Homocysteine (Hcy), thapsigargin (Thaps), acriflavine (ACF), tertiary butyl hydroquinone (tBHQ), diamide, glutathione (GSH), dithiothreitol (DTT), N-ethylmaleimide (NEM), N-acetylcysteine (NAC), L -cysteine (Cys), diphenylene iodonium (DPI), and Hoechst stain were purchased from Sigma-Aldrich.

    Techniques: Expressing

    FOXO1 Inhibition Induces HIV Reactivation in the Absence of NF-kB Recruitment via ATF4 and NFAT. a , Chromatin immunoprecipitation (ChIP) assays with antibodies against Pol II, ATF4, RelA, NFAT and IgG control at the HIV LTR, followed by qPCR using primers specific for HIV-1 LTR Nuc0 or Nuc1. Chromatin was prepared from J-Lat A2 and 5A8 cells, in which the LTR was stimulated by 1,000 nM AS1842856 treatment, 10 ng/mL TNFα or which were left untreated/DMSO. Representative experiment of at least three independent experiments. b , J-Lat cell line A58 pre-treated for 1 h with increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) before being treated with 1,000 nM AS1842856 (72 h) or 10 ng/mL TNFα (24 h). HIV-GFP reactivation was analyzed by FACS and normalized to the control. Data shown are mean ± SD of three independent experiments. c , Same experiment as in Fig. 5b , but cells were treated with increasing concentrations of Cyclosporin A (CsA), and without 1 h pre-treatment. d , Similar experiment that in Fig. 5b or Fig. 5c , but combining increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) and Cyclosporin A (CsA). Data represent average ± SD of at least three independent experiments. e , J-Lat cell line A58 was treated with increasing concentrations of Thapsigargin (0.01, 0.1, 1 µM), Brefeldin A (0.01, 0.1, 1 µg/mL) and Fenretinide (0.5, 2, 5 µM) for 24, 48 and 72 h and HIV-GFP reactivation was analyzed by FACS. f , J-Lat cell line A58 was treated with 0.5 µM Fenretinide and increasing concentrations of Ionomycin. HIV-GFP reactivation and cell viability were analyzed by FACS. Data are mean ± SD of three independent experiments. g , Model: FOXO1 inhibition leads to ER Stress. Thus, ATF4 activation through PERK and NFAT via cytosolic calcium release will promote HIV transcription and will prevent HIV latency.

    Journal: bioRxiv

    Article Title: FOXO1 promotes HIV Latency by suppressing ER stress in T cells

    doi: 10.1101/2020.04.23.058123

    Figure Lengend Snippet: FOXO1 Inhibition Induces HIV Reactivation in the Absence of NF-kB Recruitment via ATF4 and NFAT. a , Chromatin immunoprecipitation (ChIP) assays with antibodies against Pol II, ATF4, RelA, NFAT and IgG control at the HIV LTR, followed by qPCR using primers specific for HIV-1 LTR Nuc0 or Nuc1. Chromatin was prepared from J-Lat A2 and 5A8 cells, in which the LTR was stimulated by 1,000 nM AS1842856 treatment, 10 ng/mL TNFα or which were left untreated/DMSO. Representative experiment of at least three independent experiments. b , J-Lat cell line A58 pre-treated for 1 h with increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) before being treated with 1,000 nM AS1842856 (72 h) or 10 ng/mL TNFα (24 h). HIV-GFP reactivation was analyzed by FACS and normalized to the control. Data shown are mean ± SD of three independent experiments. c , Same experiment as in Fig. 5b , but cells were treated with increasing concentrations of Cyclosporin A (CsA), and without 1 h pre-treatment. d , Similar experiment that in Fig. 5b or Fig. 5c , but combining increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) and Cyclosporin A (CsA). Data represent average ± SD of at least three independent experiments. e , J-Lat cell line A58 was treated with increasing concentrations of Thapsigargin (0.01, 0.1, 1 µM), Brefeldin A (0.01, 0.1, 1 µg/mL) and Fenretinide (0.5, 2, 5 µM) for 24, 48 and 72 h and HIV-GFP reactivation was analyzed by FACS. f , J-Lat cell line A58 was treated with 0.5 µM Fenretinide and increasing concentrations of Ionomycin. HIV-GFP reactivation and cell viability were analyzed by FACS. Data are mean ± SD of three independent experiments. g , Model: FOXO1 inhibition leads to ER Stress. Thus, ATF4 activation through PERK and NFAT via cytosolic calcium release will promote HIV transcription and will prevent HIV latency.

    Article Snippet: Chemicals Cells were treated with the following compounds: FOXO inhibitor / AS1842856 (344355, Calbiochem); TNFα (300-01A, PeproTech); Raltegravir (CDS023737, Sigma-Aldrich); Prostratin (P4462, LC Laboratories); PHA-M (10576015, Sigma-Aldrich); IL-2 (I2644, Sigma-Aldrich); αCD3 (40-0038, Tonbo Biosciences); αCD28 (70-0289, Tonbo Biosciences); PMA (P8139, Sigma-Aldrich); Ionomycin (I0634, Sigma-Aldrich); PERK inhibitor II / GSK2656157 (504651, Sigma-Aldrich); GCN2 inhibitor / A-92 (2720, Axon Medchem); Imidazolo-oxindole PKR inhibitor C16 (I9785, Sigma-Aldrich); Cyclosporin A (C3662, Sigma-Aldrich); Thapsigargin (T9033, Sigma-Aldrich); Brefeldin A 1,000X Solution (00-4506-51, ThermoFisher Scientific); Fenretinide (17688, Cayman Chemicals).

    Techniques: Inhibition, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FACS, Activation Assay

    a , J-Lat cell line A58 was treated with increasing concentrations of GCN2i (A-92) in combination with 1,000 nM AS1842856 (72 h) or 10 ng/mL TNFα (24 h). In the upper panel, HIV-GFP reactivation was analyzed by FACS and relativized to the control. In the lower panel, histogram plots of percent live cells for each drug treatment are shown. b , Same experiment as in Extended Data. Fig.5a but treating cells with increasing concentrations of PKRi (Imidazolo-oxindole PKR inhibitor C16). Histogram plots representing viability are also shown. Data are represented by mean ± SD of three different donors. c , Histogram plots of percent live cells for each drug treatment are shown. Increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) (top left), Cyclosporin A (CsA) (top right), combined concentrations of PERKi and Cyclosporin A (bottom left) and increasing concentrations of Thapsigargin (0.01, 0.1, 1 µM), Brefeldin A (0.01, 0.1, 1 µg/mL) and Fenretinide (0.5, 2, 5 µM) for 24, 48 and 72 h (bottom right). Data represent mean ± SD of at least three independent experiments. d , J-Lat cell line A58 was treated with increasing concentrations of Ionomycin (0.01, 0.1, 0.5, 1 µM) for 24, 48 and 72 h and HIV-GFP reactivation (upper panel) and cell viability (lower panel) were analyzed by FACS. Data represent mean ± SD of at least three independent experiments. e , J-Lat cell line 5A8 was treated for 72 h with increasing concentrations of both Fenretinide (Y-axis) and Ionomycin (X-axis) alone or in combination and analyzed by FACS. HIV-GFP reactivation is reported as a percentage of GFP-expressing cells (% GFP+ cells) (upper panel) and viability was measured by FACS (bottom panel). Data represent average of three independent experiments.

    Journal: bioRxiv

    Article Title: FOXO1 promotes HIV Latency by suppressing ER stress in T cells

    doi: 10.1101/2020.04.23.058123

    Figure Lengend Snippet: a , J-Lat cell line A58 was treated with increasing concentrations of GCN2i (A-92) in combination with 1,000 nM AS1842856 (72 h) or 10 ng/mL TNFα (24 h). In the upper panel, HIV-GFP reactivation was analyzed by FACS and relativized to the control. In the lower panel, histogram plots of percent live cells for each drug treatment are shown. b , Same experiment as in Extended Data. Fig.5a but treating cells with increasing concentrations of PKRi (Imidazolo-oxindole PKR inhibitor C16). Histogram plots representing viability are also shown. Data are represented by mean ± SD of three different donors. c , Histogram plots of percent live cells for each drug treatment are shown. Increasing concentrations of PERKi (GSK2656157 / PERK inhibitor II) (top left), Cyclosporin A (CsA) (top right), combined concentrations of PERKi and Cyclosporin A (bottom left) and increasing concentrations of Thapsigargin (0.01, 0.1, 1 µM), Brefeldin A (0.01, 0.1, 1 µg/mL) and Fenretinide (0.5, 2, 5 µM) for 24, 48 and 72 h (bottom right). Data represent mean ± SD of at least three independent experiments. d , J-Lat cell line A58 was treated with increasing concentrations of Ionomycin (0.01, 0.1, 0.5, 1 µM) for 24, 48 and 72 h and HIV-GFP reactivation (upper panel) and cell viability (lower panel) were analyzed by FACS. Data represent mean ± SD of at least three independent experiments. e , J-Lat cell line 5A8 was treated for 72 h with increasing concentrations of both Fenretinide (Y-axis) and Ionomycin (X-axis) alone or in combination and analyzed by FACS. HIV-GFP reactivation is reported as a percentage of GFP-expressing cells (% GFP+ cells) (upper panel) and viability was measured by FACS (bottom panel). Data represent average of three independent experiments.

    Article Snippet: Chemicals Cells were treated with the following compounds: FOXO inhibitor / AS1842856 (344355, Calbiochem); TNFα (300-01A, PeproTech); Raltegravir (CDS023737, Sigma-Aldrich); Prostratin (P4462, LC Laboratories); PHA-M (10576015, Sigma-Aldrich); IL-2 (I2644, Sigma-Aldrich); αCD3 (40-0038, Tonbo Biosciences); αCD28 (70-0289, Tonbo Biosciences); PMA (P8139, Sigma-Aldrich); Ionomycin (I0634, Sigma-Aldrich); PERK inhibitor II / GSK2656157 (504651, Sigma-Aldrich); GCN2 inhibitor / A-92 (2720, Axon Medchem); Imidazolo-oxindole PKR inhibitor C16 (I9785, Sigma-Aldrich); Cyclosporin A (C3662, Sigma-Aldrich); Thapsigargin (T9033, Sigma-Aldrich); Brefeldin A 1,000X Solution (00-4506-51, ThermoFisher Scientific); Fenretinide (17688, Cayman Chemicals).

    Techniques: FACS, Expressing

    Effect of gene silencing of β1 integrin on thapsigargin-induced cell death. The indicated siRNA-transfected cells were incubated with or without 1 μM Tg or 0.2 μg/ml SubAB (Sub) or were not treated for 3 h at 37°C. GAPDH,

    Journal: Infection and Immunity

    Article Title: Identification of Subtilase Cytotoxin (SubAB) Receptors Whose Signaling, in Association with SubAB-Induced BiP Cleavage, Is Responsible for Apoptosis in HeLa Cells ▿

    doi: 10.1128/IAI.01020-10

    Figure Lengend Snippet: Effect of gene silencing of β1 integrin on thapsigargin-induced cell death. The indicated siRNA-transfected cells were incubated with or without 1 μM Tg or 0.2 μg/ml SubAB (Sub) or were not treated for 3 h at 37°C. GAPDH,

    Article Snippet: MG132 and thapsigargin (Tg) were purchased from Sigma Aldrich.

    Techniques: Transfection, Incubation

    Expression of miR-200c is regulated by Aβ-induced ER stress. (A) PC12 cells were treated with thapsigargin and cell lysate was harvested at different time points as indicated. Phospho-elF2α, CHOP, and PTEN were detected by immunoblotting (left panel). Total RNA was also extracted and subjected to qPCR analysis of miR-200c (right panel). Expression level of miR-200c is represented as the fold change compared to time point 0. (B) PC12 cells were treated with Aβ, followed by similar experiments described in (A) . Data are represented as mean ± SEM. ( * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Protective Role of microRNA-200c in Alzheimer's Disease Pathologies Is Induced by Beta Amyloid-Triggered Endoplasmic Reticulum Stress

    doi: 10.3389/fnmol.2016.00140

    Figure Lengend Snippet: Expression of miR-200c is regulated by Aβ-induced ER stress. (A) PC12 cells were treated with thapsigargin and cell lysate was harvested at different time points as indicated. Phospho-elF2α, CHOP, and PTEN were detected by immunoblotting (left panel). Total RNA was also extracted and subjected to qPCR analysis of miR-200c (right panel). Expression level of miR-200c is represented as the fold change compared to time point 0. (B) PC12 cells were treated with Aβ, followed by similar experiments described in (A) . Data are represented as mean ± SEM. ( * P

    Article Snippet: Reagents Mouse monoclonal antibodies against GAPDH and actin, rabbit polyclonal antibodies against PTEN, PERK, phosphorylated elF2 and CHOP were from Cell Signaling Technologies. β-tubulin III antibody, thapsigargin (TP) and sodium phenylbutyrate (4-PBA) were from Sigma-Aldrich.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Snapin regulates Ca 2+ efflux and influx in T cells. (A, B) Indicated cells were suspended in Ca 2+ -free medium containing 10 mM EGTA. “Control” indicates control retrovirus, whereas “Snapin” indicates that cells were infected with Snapin-encoding retrovirus. Cells were transduced with either Pep80 or C-Pep1. Cells were stained with APC-anti-Lyt2 α ' and were loaded with indo-1-AM calcium sensor dye. EGTA was added, and after 30 s (A) OKT3 or (B) thapsigargin was added. The FL5/FL4 ratio (400 nm/510 nm fluorescence emission) was monitored using a flow cytometer. (C) Cells were suspended in medium containing Ca 2+ . After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer.

    Journal: PLoS ONE

    Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

    doi: 10.1371/journal.pone.0075297

    Figure Lengend Snippet: Snapin regulates Ca 2+ efflux and influx in T cells. (A, B) Indicated cells were suspended in Ca 2+ -free medium containing 10 mM EGTA. “Control” indicates control retrovirus, whereas “Snapin” indicates that cells were infected with Snapin-encoding retrovirus. Cells were transduced with either Pep80 or C-Pep1. Cells were stained with APC-anti-Lyt2 α ' and were loaded with indo-1-AM calcium sensor dye. EGTA was added, and after 30 s (A) OKT3 or (B) thapsigargin was added. The FL5/FL4 ratio (400 nm/510 nm fluorescence emission) was monitored using a flow cytometer. (C) Cells were suspended in medium containing Ca 2+ . After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer.

    Article Snippet: The baseline fluorescence of untreated cells was measured for 30 s, the tube was removed to add 5 µ g/ml OKT3 (eBioscience, 16-0037) or 1 µ M thapsigargin (Sigma, T9033), and then flow cytometry analysis was continued.

    Techniques: Infection, Transduction, Staining, Fluorescence, Flow Cytometry, Cytometry

    STIM1L mediates slowly activating SOCE and delayed Orai1 clustering. (A) Representative Mn 2+ quench recordings (left) and quantification (right) of DKO cells co-transfected with Orai1 together with YFP–STIM1 or YFP–STIM1L ( n = 20/6/2 and 23/7/2 cells/recordings/transfections, respectively). Cells were exposed to 100 µM Mn 2+ prior to thapsigargin (Tg) addition, and fura-2 fluorescence quench was measured at 360 nm. a.u., arbitrary units. (B) TIRF images of DKO cells expressing Orai1–RFP together with YFP–STIM1 or YFP–STIM1L taken before (left), 8 min after addition of the reversible SERCA inhibitor CPA (middle) and 10 min after CPA removal and Ca 2+ re-addition (right). Insets show a threefold magnification of Orai1–RFP clusters. Scale bars: 5 µm. (C) Changes in Orai1–RFP TIRF fluorescence evoked by store depletion in cells expressing Orai1–RFP alone or together with YFP–STIM1 or YFP–STIM1L ( n = 6, 11 and 13, respectively). CPA (10 µM) was added at t = 35 s. Right panels show the delay between CPA addition and fluorescence increase, and the time to reach half-maximal fluorescence. (D) Changes in Orai1–RFP TIRF fluorescence evoked by subsequent store refilling in the cells shown in C. CPA was removed by exchanging the bath solution and 2 mM Ca 2+ was added at t = 35 s. Right, fluorescence decay time. Quantitative data show the mean±s.e.m.; * P

    Journal: Journal of Cell Science

    Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    doi: 10.1242/jcs.164228

    Figure Lengend Snippet: STIM1L mediates slowly activating SOCE and delayed Orai1 clustering. (A) Representative Mn 2+ quench recordings (left) and quantification (right) of DKO cells co-transfected with Orai1 together with YFP–STIM1 or YFP–STIM1L ( n = 20/6/2 and 23/7/2 cells/recordings/transfections, respectively). Cells were exposed to 100 µM Mn 2+ prior to thapsigargin (Tg) addition, and fura-2 fluorescence quench was measured at 360 nm. a.u., arbitrary units. (B) TIRF images of DKO cells expressing Orai1–RFP together with YFP–STIM1 or YFP–STIM1L taken before (left), 8 min after addition of the reversible SERCA inhibitor CPA (middle) and 10 min after CPA removal and Ca 2+ re-addition (right). Insets show a threefold magnification of Orai1–RFP clusters. Scale bars: 5 µm. (C) Changes in Orai1–RFP TIRF fluorescence evoked by store depletion in cells expressing Orai1–RFP alone or together with YFP–STIM1 or YFP–STIM1L ( n = 6, 11 and 13, respectively). CPA (10 µM) was added at t = 35 s. Right panels show the delay between CPA addition and fluorescence increase, and the time to reach half-maximal fluorescence. (D) Changes in Orai1–RFP TIRF fluorescence evoked by subsequent store refilling in the cells shown in C. CPA was removed by exchanging the bath solution and 2 mM Ca 2+ was added at t = 35 s. Right, fluorescence decay time. Quantitative data show the mean±s.e.m.; * P

    Article Snippet: Materials Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA).

    Techniques: Transfection, Fluorescence, Expressing

    STIM1L is poorly recruited to the plasma membrane upon store depletion yet mediates robust SOCE. (A) TIRF images of DKO MEFs expressing YFP–STIM1 and YFP–STIM1L before (left) and 10 min after addition of 1 µM thapsigargin (Tg) (right). Scale bars: 5 µm. (B) Left, percentage of plasma membrane (PM) decorated by fluorescent clusters before and after the addition of thapsigargin. Right, percentage of plasma membrane decorated by new clusters after thapsigargin addition ( n = 20/7/3 and 25/7/3 cells/recordings/transfections for YFP–STIM1 and YFP–STIM1L, respectively). (C) Ca 2+ elevations evoked by the re-addition of 2 mM Ca 2+ to cells treated with thapsigargin for 10 min in Ca 2+ -free medium (left), and quantification of thapsigargin-induced Ca 2+ influx in 2 mM Ca 2+ [right, n = 14/2/2, 35/9/6 and 51/9/6 cells/recordings/transfections for GFP–KDEL (K), YFP–STIM1 (1) or YFP–STIM1L (1L) cells, respectively]. (D) Quantification of thapsigargin-induced Ca 2+ influx in 0.5 mM Ca 2+ in cells expressing YFP–STIM1 (white bars) or YFP–STIM1L (gray bars) alone ( n = 44/13/4 and n = 57/15/4, respectively) or together with Orai1 ( n = 37/10/3 and n = 53/11/3 cells/recordings/transfections, respectively). (E) Representative changes in D1 ER ratio fluorescence measured in DKO cells co-transfected with D1 ER and either RFP–STIM1 or RFP–STIM1L. Cells were treated with 10 µM CPA for 10 min in Ca 2+ -free medium to induce store depletion, then CPA was removed by exchanging the bath solution, and 2 mM Ca 2+ was added 3 min later to promote store refilling (left). Right, statistical evaluation of ER refilling velocity upon 0.5 mM or 2 mM Ca 2+ re-addition ( n = 25/11/3, 25/12/3 cells/recordings/transfections at 0.5 mM and 17/7/3, 17/9/3 cells/recordings/transfections at 2 mM for RFP–STIM1 or RFP–STIM1L cells, respectively). Data show the mean±s.e.m.; * P

    Journal: Journal of Cell Science

    Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    doi: 10.1242/jcs.164228

    Figure Lengend Snippet: STIM1L is poorly recruited to the plasma membrane upon store depletion yet mediates robust SOCE. (A) TIRF images of DKO MEFs expressing YFP–STIM1 and YFP–STIM1L before (left) and 10 min after addition of 1 µM thapsigargin (Tg) (right). Scale bars: 5 µm. (B) Left, percentage of plasma membrane (PM) decorated by fluorescent clusters before and after the addition of thapsigargin. Right, percentage of plasma membrane decorated by new clusters after thapsigargin addition ( n = 20/7/3 and 25/7/3 cells/recordings/transfections for YFP–STIM1 and YFP–STIM1L, respectively). (C) Ca 2+ elevations evoked by the re-addition of 2 mM Ca 2+ to cells treated with thapsigargin for 10 min in Ca 2+ -free medium (left), and quantification of thapsigargin-induced Ca 2+ influx in 2 mM Ca 2+ [right, n = 14/2/2, 35/9/6 and 51/9/6 cells/recordings/transfections for GFP–KDEL (K), YFP–STIM1 (1) or YFP–STIM1L (1L) cells, respectively]. (D) Quantification of thapsigargin-induced Ca 2+ influx in 0.5 mM Ca 2+ in cells expressing YFP–STIM1 (white bars) or YFP–STIM1L (gray bars) alone ( n = 44/13/4 and n = 57/15/4, respectively) or together with Orai1 ( n = 37/10/3 and n = 53/11/3 cells/recordings/transfections, respectively). (E) Representative changes in D1 ER ratio fluorescence measured in DKO cells co-transfected with D1 ER and either RFP–STIM1 or RFP–STIM1L. Cells were treated with 10 µM CPA for 10 min in Ca 2+ -free medium to induce store depletion, then CPA was removed by exchanging the bath solution, and 2 mM Ca 2+ was added 3 min later to promote store refilling (left). Right, statistical evaluation of ER refilling velocity upon 0.5 mM or 2 mM Ca 2+ re-addition ( n = 25/11/3, 25/12/3 cells/recordings/transfections at 0.5 mM and 17/7/3, 17/9/3 cells/recordings/transfections at 2 mM for RFP–STIM1 or RFP–STIM1L cells, respectively). Data show the mean±s.e.m.; * P

    Article Snippet: Materials Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA).

    Techniques: Expressing, Transfection, Fluorescence

    STIM1L does not enlarge plasma membrane clusters upon store depletion. (A) TIRF images of cells expressing YFP–STIM1, YFP–STIM1L and STIM1LΔABD taken before (left) and 10 min after thapsigargin (Tg) addition (right). Insets, a threefold magnification to show the morphology of fluorescent clusters. Scale bars: 5 µm. (B) Quantitative analysis of TIRF images showing the effects of thapsigargin on the density, size and intensity of YFP clusters. Right panels show the absolute increase in cluster density, size and intensity following thapsigargin addition ( n = 20/7/3, 25/7/3 and 34/7/3 cells/recordings/transfections for YFP–STIM1, YFP–STIM1L and STIM1LΔABD. A.U., arbitrary units. Data show the mean±s.e.m.; * P

    Journal: Journal of Cell Science

    Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    doi: 10.1242/jcs.164228

    Figure Lengend Snippet: STIM1L does not enlarge plasma membrane clusters upon store depletion. (A) TIRF images of cells expressing YFP–STIM1, YFP–STIM1L and STIM1LΔABD taken before (left) and 10 min after thapsigargin (Tg) addition (right). Insets, a threefold magnification to show the morphology of fluorescent clusters. Scale bars: 5 µm. (B) Quantitative analysis of TIRF images showing the effects of thapsigargin on the density, size and intensity of YFP clusters. Right panels show the absolute increase in cluster density, size and intensity following thapsigargin addition ( n = 20/7/3, 25/7/3 and 34/7/3 cells/recordings/transfections for YFP–STIM1, YFP–STIM1L and STIM1LΔABD. A.U., arbitrary units. Data show the mean±s.e.m.; * P

    Article Snippet: Materials Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA).

    Techniques: Expressing, Transfection

    Cluster expansion requires the STIM1 lysine-rich tail but not cytosolic Ca 2+ elevations. (A) Left, TIRF images of cells expressing YFP–STIM1 and YFP–STIM1L taken before (left) and 10 min after thapsigargin (Tg) addition (right) in conditions preventing cytosolic Ca 2+ elevations (20 µM BAPTA-AM and 50 µM La 3+ ). Insets, a 2.6-fold magnification to show the morphology of fluorescent clusters. Scale bars: 10 µm. Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. ( n = 33/5/3, 38/5/3, 36/5/3 and 44/5/3 cells/recordings/transfections). (B) Left, TIRF images of cells expressing YFP–STIM1ΔK and YFP–STIM1LΔK taken before (left) and 10 min after thapsigargin addition (right). Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. ( n = 41/7/6, 49/6/5, 51/7/5 and 42/6/5 cells/recordings/transfections). Quantitative data show the mean±s.e.m.; * P

    Journal: Journal of Cell Science

    Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    doi: 10.1242/jcs.164228

    Figure Lengend Snippet: Cluster expansion requires the STIM1 lysine-rich tail but not cytosolic Ca 2+ elevations. (A) Left, TIRF images of cells expressing YFP–STIM1 and YFP–STIM1L taken before (left) and 10 min after thapsigargin (Tg) addition (right) in conditions preventing cytosolic Ca 2+ elevations (20 µM BAPTA-AM and 50 µM La 3+ ). Insets, a 2.6-fold magnification to show the morphology of fluorescent clusters. Scale bars: 10 µm. Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. ( n = 33/5/3, 38/5/3, 36/5/3 and 44/5/3 cells/recordings/transfections). (B) Left, TIRF images of cells expressing YFP–STIM1ΔK and YFP–STIM1LΔK taken before (left) and 10 min after thapsigargin addition (right). Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. ( n = 41/7/6, 49/6/5, 51/7/5 and 42/6/5 cells/recordings/transfections). Quantitative data show the mean±s.e.m.; * P

    Article Snippet: Materials Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA).

    Techniques: Expressing, Transfection

    STIM1L does not recruit cortical ER cisternae upon store depletion. (A) Ultra-structural analysis of DKO cells expressing pCDNA, YFP–STIM1 or YFP–STIM1L before (top) or 10 min after exposure to 1 µM thapsigargin (Tg) (bottom). Images show sheets of cER (asterisks) apposed to the plasma membrane (closed circle). Arrowhead denotes the dish bottom; θ marks an adjacent cell. Scale bar: 100 nm. (B,C) Percentage of plasma membrane (PM) decorated by cER in DKO cells (B) and myoblasts (C) before and after addition of thapsigargin ( n = 71–102 for each condition from at least three independent experiments). (D) Quantification of the Ca 2+ elevations evoked by Ca 2+ re-addition to thapsigargin-treated myoblasts expressing GFP–KDEL, YFP–STIM1 or YFP–STIM1L ( n = 25/5/3, 27/5/3 and 26/5/3 cells/recordings/transfections, respectively) Quantitative data show the mean±s.e.m.; ** P

    Journal: Journal of Cell Science

    Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    doi: 10.1242/jcs.164228

    Figure Lengend Snippet: STIM1L does not recruit cortical ER cisternae upon store depletion. (A) Ultra-structural analysis of DKO cells expressing pCDNA, YFP–STIM1 or YFP–STIM1L before (top) or 10 min after exposure to 1 µM thapsigargin (Tg) (bottom). Images show sheets of cER (asterisks) apposed to the plasma membrane (closed circle). Arrowhead denotes the dish bottom; θ marks an adjacent cell. Scale bar: 100 nm. (B,C) Percentage of plasma membrane (PM) decorated by cER in DKO cells (B) and myoblasts (C) before and after addition of thapsigargin ( n = 71–102 for each condition from at least three independent experiments). (D) Quantification of the Ca 2+ elevations evoked by Ca 2+ re-addition to thapsigargin-treated myoblasts expressing GFP–KDEL, YFP–STIM1 or YFP–STIM1L ( n = 25/5/3, 27/5/3 and 26/5/3 cells/recordings/transfections, respectively) Quantitative data show the mean±s.e.m.; ** P

    Article Snippet: Materials Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA).

    Techniques: Expressing, Transfection

    Treatments with deoxysphingoid bases cause depletion of ER Ca 2+ and mitochondrial Ca 2+ loading in neurons. (A) A typical trace (from a MN culture) indicating how thapsigargin and ionomycin were used to estimate ER and mitochondrial Ca 2+ levels. (B) At 5–8 DIV, MNs were treated with either vehicle control (ethanol) or the sphingoid bases for 24 h prior to live cell imaging. Thapsigargin was used to estimate ER Ca 2+ . Average ER Ca 2+ was established from 42 to 55 cells per condition, from 4 to 5 independent experiments. ( P

    Journal: Neurobiology of Disease

    Article Title: Hereditary sensory neuropathy type 1-associated deoxysphingolipids cause neurotoxicity, acute calcium handling abnormalities and mitochondrial dysfunction in vitro

    doi: 10.1016/j.nbd.2018.05.008

    Figure Lengend Snippet: Treatments with deoxysphingoid bases cause depletion of ER Ca 2+ and mitochondrial Ca 2+ loading in neurons. (A) A typical trace (from a MN culture) indicating how thapsigargin and ionomycin were used to estimate ER and mitochondrial Ca 2+ levels. (B) At 5–8 DIV, MNs were treated with either vehicle control (ethanol) or the sphingoid bases for 24 h prior to live cell imaging. Thapsigargin was used to estimate ER Ca 2+ . Average ER Ca 2+ was established from 42 to 55 cells per condition, from 4 to 5 independent experiments. ( P

    Article Snippet: Relative ER Ca2+ concentration and mitochondrial Ca2+ concentration were measured in Ca2+ -free recording medium containing 0.5 mM EGTA, first using thapsigargin (10 μM, Sigma) to estimate ER Ca2+ , followed by ionomycin treatment (10 μM, Sigma), to estimate mitochondrial Ca2+ ( ).

    Techniques: Live Cell Imaging

    Dexamethasone stimulates intracellular Ca2+ release and SOCE and co-treatment with dexamethasone and SOC inhibitors markedly enhances ALL cells death Cells were loaded with Fura-2/AM, and the changes in intracellular Ca 2+ , [Ca 2+ ]i, (F340/F380) were monitored. (A, B) The colored time-lapse images and the graphical representation show the changes in [Ca 2+ ]i evoked by dexamethasone (Dex), in Reh and Nalm-6 cell lines, respectively, in Ca 2+ -containing and in Ca 2+ -free buffer. (C, D) ALL cells before exposure to 100 nM dexamethasone (Dex) in Ca 2+ buffer were preincubated (20 minutes) with U73122 (10 μM), SKF96365 (10 μM) or 2-APB (10 μM). Data are mean ± SEM (n = 5). (E) Effect of dexamethasone (Dex) on SOCE activation in ALL cells. ALL cells ER calcium stores were depleted with thapsigargin (TG, 1 μM) in calcium-free suspension medium in the presence or absence of 2-APB (10 μM), cells were then treated without or with 100 nM dexamethasone, followed by addition of 1.8 mM CaCl 2 . Data are mean ± SEM (n = 3). Reh (F) and Nalm-6 (G) cells were treated with SOC inhibitors (SKF 96365, 5 μM and 2-APB, 5 μM) and dexamethasone (100 nM) alone or in combination for 48 h. Cell death was detected by MTT metabolic colorimetric assay. Data are representative of triplicate experiments. *p

    Journal: Oncotarget

    Article Title: Improvement of dexamethasone sensitivity by chelation of intracellular Ca2+ in pediatric acute lymphoblastic leukemia cells through the prosurvival kinase ERK1/2 deactivation

    doi: 10.18632/oncotarget.16039

    Figure Lengend Snippet: Dexamethasone stimulates intracellular Ca2+ release and SOCE and co-treatment with dexamethasone and SOC inhibitors markedly enhances ALL cells death Cells were loaded with Fura-2/AM, and the changes in intracellular Ca 2+ , [Ca 2+ ]i, (F340/F380) were monitored. (A, B) The colored time-lapse images and the graphical representation show the changes in [Ca 2+ ]i evoked by dexamethasone (Dex), in Reh and Nalm-6 cell lines, respectively, in Ca 2+ -containing and in Ca 2+ -free buffer. (C, D) ALL cells before exposure to 100 nM dexamethasone (Dex) in Ca 2+ buffer were preincubated (20 minutes) with U73122 (10 μM), SKF96365 (10 μM) or 2-APB (10 μM). Data are mean ± SEM (n = 5). (E) Effect of dexamethasone (Dex) on SOCE activation in ALL cells. ALL cells ER calcium stores were depleted with thapsigargin (TG, 1 μM) in calcium-free suspension medium in the presence or absence of 2-APB (10 μM), cells were then treated without or with 100 nM dexamethasone, followed by addition of 1.8 mM CaCl 2 . Data are mean ± SEM (n = 3). Reh (F) and Nalm-6 (G) cells were treated with SOC inhibitors (SKF 96365, 5 μM and 2-APB, 5 μM) and dexamethasone (100 nM) alone or in combination for 48 h. Cell death was detected by MTT metabolic colorimetric assay. Data are representative of triplicate experiments. *p

    Article Snippet: Fura-2/AM, Rhod-2/AM, Bapta-AM, U73122, SKF96365 and thapsigargin were purchased from Abcam Biochemicals, France.

    Techniques: Activation Assay, MTT Assay, Colorimetric Assay

    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

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

    Journal: Frontiers in Cellular Neuroscience

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

    doi: 10.3389/fncel.2014.00016

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.18595

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

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

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