thapsigargin thap  (Millipore)


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    Thapsigargin
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    Catalog Number:
    t9033
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    Structured Review

    Millipore thapsigargin thap
    Thapsigargin

    https://www.bioz.com/result/thapsigargin thap/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thapsigargin thap - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells"

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

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-148

    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
    Figure Legend 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

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

    2) Product Images from "Serotonin reuptake inhibitors act centrally to cause bone loss in mice by counteracting a local antiresorptive effect"

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

    Journal: Nature medicine

    doi: 10.1038/nm.4166

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

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

    Related Articles

    Concentration Assay:

    Article Title: Inhibitors of eIF2? Dephosphorylation Slow Replication and Stabilize Latency in Toxoplasma gondii
    Article Snippet: .. Filter-purified parasites were incubated in DMEM plus 1% FBS supplemented with 10 μM thapsigargin (T9033; Sigma) or vehicle (DMSO) for 1 h. Following a wash step, parasites were incubated for another hour in DMEM plus 1% FBS supplemented with the indicated concentration of SAL, GA, or vehicle (DMSO). .. All incubation steps were carried out at 37°C and 5% CO2 .

    Article Title: Enhanced Astrocytic Nitric Oxide Production and Neuronal Modifications in the Neocortex of a NOS2 Mutant Mouse
    Article Snippet: .. The following drugs were added to the incubating solution at various time intervals before imaging, and the same concentration was maintained in the superfusing solution during imaging, unless noted otherwise: Nw -Nitro-L-arginine (L-NNA); L–N6- (1–iminoethyl) lysine, 2HCl (L-NIL); N-nitro-L-arginine methyl ester (L-NAME); Thapsigargin, all purchased from Sigma-Aldrich, Israel, and N-[[3 (aminomethyl) phenyl]methyl]ethanimidamide dihydrochloride (1400W) was purchased from Tocris (Bristol, UK). .. Western Blot Analysis The neocortex was dissected immediately following decapitation, homogenized in ice-cold lysis buffer, and centrifuged at 14,000 rpm for 15 min.

    Incubation:

    Article Title: Inhibitors of eIF2? Dephosphorylation Slow Replication and Stabilize Latency in Toxoplasma gondii
    Article Snippet: .. Filter-purified parasites were incubated in DMEM plus 1% FBS supplemented with 10 μM thapsigargin (T9033; Sigma) or vehicle (DMSO) for 1 h. Following a wash step, parasites were incubated for another hour in DMEM plus 1% FBS supplemented with the indicated concentration of SAL, GA, or vehicle (DMSO). .. All incubation steps were carried out at 37°C and 5% CO2 .

    other:

    Article Title: A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic ? cells
    Article Snippet: Sources of reagents and application of test substances Exendin-4, forskolin, caffeine, ryanodine, and thapsigargin were from Sigma-Aldrich.

    Article Title: Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL
    Article Snippet: Materials The 4-phenylbutyric acid (PBA) compound, N-acetylcystein (NAC) and thapsigargin were obtained from Sigma-Aldrich (St. Louis, MO).

    Article Title: cAMP-dependent Mobilization of Intracellular Ca2+ Stores by Activation of Ryanodine Receptors in Pancreatic β-Cells
    Article Snippet: 8-Br-cAMP, nimodipine, ryanodine, caffeine, and thapsigargin were from Sigma. ( R p)-cAMPS was from BioLog Life Sciences Institute (Bremen, Germany).

    Article Title: TRPA1 and TRPV1 are required for lidocaine-evoked calcium influx and neuropeptide release but not cytotoxicity in mouse sensory neurons
    Article Snippet: For capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide), BCTC (4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide,), HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide) stock solutions were prepared in absolute ethanol, while for allyl isothiocyanate (AITC) and thapsigargin, and dimethyl sulfoxide was used (all compounds from Sigma-Aldrich).

    Imaging:

    Article Title: Enhanced Astrocytic Nitric Oxide Production and Neuronal Modifications in the Neocortex of a NOS2 Mutant Mouse
    Article Snippet: .. The following drugs were added to the incubating solution at various time intervals before imaging, and the same concentration was maintained in the superfusing solution during imaging, unless noted otherwise: Nw -Nitro-L-arginine (L-NNA); L–N6- (1–iminoethyl) lysine, 2HCl (L-NIL); N-nitro-L-arginine methyl ester (L-NAME); Thapsigargin, all purchased from Sigma-Aldrich, Israel, and N-[[3 (aminomethyl) phenyl]methyl]ethanimidamide dihydrochloride (1400W) was purchased from Tocris (Bristol, UK). .. Western Blot Analysis The neocortex was dissected immediately following decapitation, homogenized in ice-cold lysis buffer, and centrifuged at 14,000 rpm for 15 min.

    Cell Culture:

    Article Title: ?-Adrenergic Receptor Stimulation Induces Endoplasmic Reticulum Stress in Adult Cardiac Myocytes: Role in Apoptosis
    Article Snippet: .. ARVMs, cultured for 24 h, were treated with isoproterenol (ISO; 10 μM; Sigma), forskolin (10 μM; Sigma), thapsigargin (2 μM; Sigma) or brefeldin A (1 μM; Sigma) for 15 min, 3 h or 24 h. For treatment with ISO, dishes were supplemented with ascorbic acid (100 μM). .. CGP20712A (0.3 μM; Sigma), ICI 118551 (0.1 μM; Sigma), salubrinal (1 or 10 μM) or z-ATAD-FMK (10 μM) were added for 30 min prior to ISO treatment.

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    Millipore thapsigargin thaps
    Astrocytes are resistant to ER stress-induced cell death. (A) Primary astrocytes, NPCs, and microglia were treated with <t>Thaps</t> (0.03 to 3 μM) or Tunic (0.1 to 10 μM) for 24 h followed by assessment of cell viability by WST-1 assay. (B)
    Thapsigargin Thaps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thapsigargin thaps/product/Millipore
    Average 99 stars, based on 365 article reviews
    Price from $9.99 to $1999.99
    thapsigargin thaps - by Bioz Stars, 2020-09
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    91
    Millipore thap
    Astrocytes are resistant to ER stress-induced cell death. (A) Primary astrocytes, NPCs, and microglia were treated with <t>Thaps</t> (0.03 to 3 μM) or Tunic (0.1 to 10 μM) for 24 h followed by assessment of cell viability by WST-1 assay. (B)
    Thap, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thap/product/Millipore
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thap - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Astrocytes are resistant to ER stress-induced cell death. (A) Primary astrocytes, NPCs, and microglia were treated with Thaps (0.03 to 3 μM) or Tunic (0.1 to 10 μM) for 24 h followed by assessment of cell viability by WST-1 assay. (B)

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: Astrocytes are resistant to ER stress-induced cell death. (A) Primary astrocytes, NPCs, and microglia were treated with Thaps (0.03 to 3 μM) or Tunic (0.1 to 10 μM) for 24 h followed by assessment of cell viability by WST-1 assay. (B)

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: WST-1 Assay

    ER stress stimulates JAK1-dependent STAT3 activation. Astrocytes (A) or the pancreatic β-cell line INS832/13 (C) was treated with Thaps (1 μM) for the indicated times followed by immunoblotting. (B) NPCs were treated with the indicated

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: ER stress stimulates JAK1-dependent STAT3 activation. Astrocytes (A) or the pancreatic β-cell line INS832/13 (C) was treated with Thaps (1 μM) for the indicated times followed by immunoblotting. (B) NPCs were treated with the indicated

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Activation Assay

    JAK1 and NF-κB contribute to inflammatory gene expression. (A) Astrocytes were transfected with control or JAK1 siRNA (50 pmol/ml) for 48 h. Cells were then treated with Thaps (1 μM) for the indicated times followed by immunoblotting for

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: JAK1 and NF-κB contribute to inflammatory gene expression. (A) Astrocytes were transfected with control or JAK1 siRNA (50 pmol/ml) for 48 h. Cells were then treated with Thaps (1 μM) for the indicated times followed by immunoblotting for

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Expressing, Transfection

    ER stress stimulates an inflammatory response. (A) Astrocytes were treated with Thaps (1 μM) for the indicated times followed by analysis of IL-6, CCL2, and CCL20 mRNA by qRT-PCR. (B) Astrocytes were exposed transiently to Thaps (1 μM,

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: ER stress stimulates an inflammatory response. (A) Astrocytes were treated with Thaps (1 μM) for the indicated times followed by analysis of IL-6, CCL2, and CCL20 mRNA by qRT-PCR. (B) Astrocytes were exposed transiently to Thaps (1 μM,

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Quantitative RT-PCR

    ER stress-induced JAK/STAT signaling is PERK dependent. (A) Astrocytes were transfected with control, PERK, IRE1, or ATF6 siRNA (50 pmol/ml) for 48 h. Cells were then treated with Thaps (1 μM, 4 h), and the levels of CCL2 and IL-6 mRNA were measured

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: ER stress-induced JAK/STAT signaling is PERK dependent. (A) Astrocytes were transfected with control, PERK, IRE1, or ATF6 siRNA (50 pmol/ml) for 48 h. Cells were then treated with Thaps (1 μM, 4 h), and the levels of CCL2 and IL-6 mRNA were measured

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Transfection

    ER stress drives JAK-dependent gene expression. (A) Astrocytes were treated with Thaps (1 μM, 4 h) in the absence or presence of P6 (0.5 μM) or AZD1480 (1.0 μM), followed by analysis of IL-6, CCL2, and CHOP mRNA by qRT-PCR. (B)

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: ER stress drives JAK-dependent gene expression. (A) Astrocytes were treated with Thaps (1 μM, 4 h) in the absence or presence of P6 (0.5 μM) or AZD1480 (1.0 μM), followed by analysis of IL-6, CCL2, and CHOP mRNA by qRT-PCR. (B)

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Expressing, Quantitative RT-PCR

    ER-stressed astrocytes stimulate a PERK-dependent microglia-astrocyte feed-forward inflammatory loop. (A) Astrocytes were transfected with control or PERK siRNA (50 pmol/ml) for 48 h and then exposed transiently to Thaps (1 μM, 2 h); the cells

    Journal: Molecular and Cellular Biology

    Article Title: PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

    doi: 10.1128/MCB.00980-14

    Figure Lengend Snippet: ER-stressed astrocytes stimulate a PERK-dependent microglia-astrocyte feed-forward inflammatory loop. (A) Astrocytes were transfected with control or PERK siRNA (50 pmol/ml) for 48 h and then exposed transiently to Thaps (1 μM, 2 h); the cells

    Article Snippet: Thapsigargin (Thaps), tunicamycin, and pyridone 6 (P6; JAK inhibitor I) were from Calbiochem.

    Techniques: Transfection

    ER stressed astrocytes secrete a molecule(s) which upregulate ER stress responses in astrocytes and neurons. (A) Outline for the astrocyte conditioned media (ACM) experiments. Following treatment with the vehicle (DMSO) or 1.0 μM thapsigargin (Thaps) for 2 h, primary murine astrocyte cultures were washed with PBS three times, then incubated in fresh media for 24 h. After this period, the media (ACM) was collected. Lactate dehydrogenase (LDH) activity in the collected ACM was measured using the LDH assay. N = 3, p = 0.7 determined by Mann Whitney test. Not significant (n.s.) ( B-C ) Primary murine astrocytes (B) and murine HT-22 hippocampal neurons (C) incubated with control (UT ACM) or ER stressed ACM (Thaps ACM) for 6 h. CHOP and GRP78 mRNA expression was quantified using RT-qPCR. Immunoblot analysis was used to examine GRP78, spliced XBP-1 (sXBP-1) and CHOP protein expression. 1.0 μM ( B ) or 0.1 μM ( C ) Thaps was used as the positive control for B and C, respectively. GAPDH was used as the loading control. N = 3 independent cell culture preparations, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test. (D) HT-22 neurons incubated with control or ER stressed ACM from tunicamycin (Tunic)-treated primary murine astrocytes for the indicated time points. Immunoblot analysis was used to examine GRP78, ATF4 and CHOP protein expression. GAPDH was used as the loading control.

    Journal: Journal of neurochemistry

    Article Title: Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system

    doi: 10.1111/jnc.14642

    Figure Lengend Snippet: ER stressed astrocytes secrete a molecule(s) which upregulate ER stress responses in astrocytes and neurons. (A) Outline for the astrocyte conditioned media (ACM) experiments. Following treatment with the vehicle (DMSO) or 1.0 μM thapsigargin (Thaps) for 2 h, primary murine astrocyte cultures were washed with PBS three times, then incubated in fresh media for 24 h. After this period, the media (ACM) was collected. Lactate dehydrogenase (LDH) activity in the collected ACM was measured using the LDH assay. N = 3, p = 0.7 determined by Mann Whitney test. Not significant (n.s.) ( B-C ) Primary murine astrocytes (B) and murine HT-22 hippocampal neurons (C) incubated with control (UT ACM) or ER stressed ACM (Thaps ACM) for 6 h. CHOP and GRP78 mRNA expression was quantified using RT-qPCR. Immunoblot analysis was used to examine GRP78, spliced XBP-1 (sXBP-1) and CHOP protein expression. 1.0 μM ( B ) or 0.1 μM ( C ) Thaps was used as the positive control for B and C, respectively. GAPDH was used as the loading control. N = 3 independent cell culture preparations, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test. (D) HT-22 neurons incubated with control or ER stressed ACM from tunicamycin (Tunic)-treated primary murine astrocytes for the indicated time points. Immunoblot analysis was used to examine GRP78, ATF4 and CHOP protein expression. GAPDH was used as the loading control.

    Article Snippet: Thapsigargin (Thaps; 586005–1MG) and tunicamycin (65–438-010MG) were from Calbiochem.

    Techniques: Incubation, Activity Assay, Lactate Dehydrogenase Assay, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Positive Control, Cell Culture

    The stress factor(s) secreted by ER stressed astrocytes is not a microparticle or proteinase K-sensitive protein. ( A ) Murine HT-22 hippocampal neurons were stimulated with 10 ng/mL of IL-1β, IL-6 or TNFα for 6 h. CHOP and GRP78 mRNA expression was quantified using RT-qPCR. Thaps (1μM) was used as a positive control. ( B ) Microparticles (MP) were isolated from the ER stressed astrocyte conditioned media (ACM) via ultracentrifugation (160,000 × g for 1 h). The supernatant (sup) was collected, and the pellet at the bottom of the tube (contains MPs) was resuspended in PBS. The centrifuged ACM (thapsigargin (Thaps) ACM Sup) and the solution containing the MPs (Thaps ACM MP pellet) were incubated with astrocytes for 6 h. ATF4, CHOP and GRP78 mRNA was quantified using RT-qPCR. ( C ) ER stressed ACM was treated with immobilized proteinase K for 1 h at 37°C. After which, the ACM was centrifuged at 1000 × g for 2 min to separate the beads and the media. The proteinase K-free ACM was collected, then incubated with astrocytes for 4 h. CHOP and GRP78 mRNA was quantified using RT-qPCR. To confirm that proteinase K was functional, 1 mg/mL of BSA was treated with proteinase K for 1 h at 37°C. Proteins from the BSA (lanes 1 and 2) or proteinase K-treated BSA (lanes 3 and 4) solutions were stained using Coomassie Blue staining following SDS-PAGE. A B (N = 3), C (N = 4) independent cell culture preparations, ***P ≤ 0.001, **** P ≤ 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test. ( D ) ACM from control or Thaps treated astrocytes was filtered through a 100 kDa molecular weight cut off filter. In a separate sample, Thaps was added directly to the ACM prior to filtration. The filtrate (F) or retentate (R) were then added to HT-22 cells (final concentration of 50% ACM) for 18 h followed by immunoblotting. ( E ) Astrocytes were cultured in media with serum, without serum or without serum + 100 ug/ml BSA to produce ACM from UT or Thaps treated astrocytes. The media was incubated with HT-22 cells (final concentration of 50% ACM) for 18h followed by immunoblotting. ( F ) ACM was incubated at RT for 1 h without or with Cleanascite, then centrifuged at 2000 × g and added to HT-22 cells (final concentration of 50% ACM) for 18h followed by immunoblotting. (G) NCM was incubated at RT for 1 h without or with Cleanascite, then centrifuged at 2000 × g and added to astrocytes for 18h followed by immunoblotting.

    Journal: Journal of neurochemistry

    Article Title: Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system

    doi: 10.1111/jnc.14642

    Figure Lengend Snippet: The stress factor(s) secreted by ER stressed astrocytes is not a microparticle or proteinase K-sensitive protein. ( A ) Murine HT-22 hippocampal neurons were stimulated with 10 ng/mL of IL-1β, IL-6 or TNFα for 6 h. CHOP and GRP78 mRNA expression was quantified using RT-qPCR. Thaps (1μM) was used as a positive control. ( B ) Microparticles (MP) were isolated from the ER stressed astrocyte conditioned media (ACM) via ultracentrifugation (160,000 × g for 1 h). The supernatant (sup) was collected, and the pellet at the bottom of the tube (contains MPs) was resuspended in PBS. The centrifuged ACM (thapsigargin (Thaps) ACM Sup) and the solution containing the MPs (Thaps ACM MP pellet) were incubated with astrocytes for 6 h. ATF4, CHOP and GRP78 mRNA was quantified using RT-qPCR. ( C ) ER stressed ACM was treated with immobilized proteinase K for 1 h at 37°C. After which, the ACM was centrifuged at 1000 × g for 2 min to separate the beads and the media. The proteinase K-free ACM was collected, then incubated with astrocytes for 4 h. CHOP and GRP78 mRNA was quantified using RT-qPCR. To confirm that proteinase K was functional, 1 mg/mL of BSA was treated with proteinase K for 1 h at 37°C. Proteins from the BSA (lanes 1 and 2) or proteinase K-treated BSA (lanes 3 and 4) solutions were stained using Coomassie Blue staining following SDS-PAGE. A B (N = 3), C (N = 4) independent cell culture preparations, ***P ≤ 0.001, **** P ≤ 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test. ( D ) ACM from control or Thaps treated astrocytes was filtered through a 100 kDa molecular weight cut off filter. In a separate sample, Thaps was added directly to the ACM prior to filtration. The filtrate (F) or retentate (R) were then added to HT-22 cells (final concentration of 50% ACM) for 18 h followed by immunoblotting. ( E ) Astrocytes were cultured in media with serum, without serum or without serum + 100 ug/ml BSA to produce ACM from UT or Thaps treated astrocytes. The media was incubated with HT-22 cells (final concentration of 50% ACM) for 18h followed by immunoblotting. ( F ) ACM was incubated at RT for 1 h without or with Cleanascite, then centrifuged at 2000 × g and added to HT-22 cells (final concentration of 50% ACM) for 18h followed by immunoblotting. (G) NCM was incubated at RT for 1 h without or with Cleanascite, then centrifuged at 2000 × g and added to astrocytes for 18h followed by immunoblotting.

    Article Snippet: Thapsigargin (Thaps; 586005–1MG) and tunicamycin (65–438-010MG) were from Calbiochem.

    Techniques: Expressing, Quantitative RT-PCR, Positive Control, Isolation, Incubation, Functional Assay, Staining, SDS Page, Cell Culture, Molecular Weight, Filtration, Concentration Assay

    Mild exposure to the stress factor(s) promotes an adaptive state in neurons. ( A ) Murine HT-22 hippocampal neurons were stimulated with control or ER stressed astrocyte conditioned media (UT or Thaps ACM) for 6 or 24 h. Caspase-3 activity was assessed by measuring the fluorescence intensity of the cleaved capase-3 substrate Ac-DEVD-AMC. Caspase-3 activity was expressed as arbitrary fluorescence units (AU). N = 3, *P ≤ 0.05 determined by one-way ANOVA with Tukey’s multiple comparisons test. ( B ) HT-22 neurons were treated with astrocyte conditioned media (UT or Thaps ACM) for 24 h followed by measuring phosphatidylserine externalization using a split luciferase coupled to Annexin V (Real-time Glo), data are expressed as arbitrary luminescent units (AU). N = 3 independent cell culture preparations, *P ≤ 0.05 determined by Student’s T test. ( C ) HT-22 neurons incubated with 25% control or ER stressed ACM for the indicated time points. GRP78 and CHOP protein expression was examined using immunoblot analysis. ( D ) HT-22 neurons incubated with 25% control or ER stressed ACM for 2 or 6 h, followed with a 4 or 24 h washout period. GRP78 and CHOP protein expression was examined using immunoblot analysis. GAPDH was used as the loading control. UT, untreated; UTA, control ACM; TA, Thaps ACM.

    Journal: Journal of neurochemistry

    Article Title: Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system

    doi: 10.1111/jnc.14642

    Figure Lengend Snippet: Mild exposure to the stress factor(s) promotes an adaptive state in neurons. ( A ) Murine HT-22 hippocampal neurons were stimulated with control or ER stressed astrocyte conditioned media (UT or Thaps ACM) for 6 or 24 h. Caspase-3 activity was assessed by measuring the fluorescence intensity of the cleaved capase-3 substrate Ac-DEVD-AMC. Caspase-3 activity was expressed as arbitrary fluorescence units (AU). N = 3, *P ≤ 0.05 determined by one-way ANOVA with Tukey’s multiple comparisons test. ( B ) HT-22 neurons were treated with astrocyte conditioned media (UT or Thaps ACM) for 24 h followed by measuring phosphatidylserine externalization using a split luciferase coupled to Annexin V (Real-time Glo), data are expressed as arbitrary luminescent units (AU). N = 3 independent cell culture preparations, *P ≤ 0.05 determined by Student’s T test. ( C ) HT-22 neurons incubated with 25% control or ER stressed ACM for the indicated time points. GRP78 and CHOP protein expression was examined using immunoblot analysis. ( D ) HT-22 neurons incubated with 25% control or ER stressed ACM for 2 or 6 h, followed with a 4 or 24 h washout period. GRP78 and CHOP protein expression was examined using immunoblot analysis. GAPDH was used as the loading control. UT, untreated; UTA, control ACM; TA, Thaps ACM.

    Article Snippet: Thapsigargin (Thaps; 586005–1MG) and tunicamycin (65–438-010MG) were from Calbiochem.

    Techniques: Activity Assay, Fluorescence, Luciferase, Cell Culture, Incubation, Expressing

    Neuronal ER stress promotes PERK-dependent inflammatory signaling in astrocytes. (A) Astrocytes were transfected with non-targeting (control) or PERK siRNA for 48 h followed by treatment with thaps (1 μM) for 60 min. Cell lysates were then immunoblotted for the indicated protiens. ( B) Astrocytes were transfected as in A for 48 h prior to incubating with control or ER stressed HT-22 conditioned media (UT or Thaps NCM) for 6 h. IL-6, CCL2 and CCL20 mRNA expression was quantified using RT-qPCR. N = 3 independent cell culture preparations, **P ≤ 0.01, *** P ≤ 0.001 determined by two-way ANOVA with Bonferroni’s multiple comparisons test.

    Journal: Journal of neurochemistry

    Article Title: Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system

    doi: 10.1111/jnc.14642

    Figure Lengend Snippet: Neuronal ER stress promotes PERK-dependent inflammatory signaling in astrocytes. (A) Astrocytes were transfected with non-targeting (control) or PERK siRNA for 48 h followed by treatment with thaps (1 μM) for 60 min. Cell lysates were then immunoblotted for the indicated protiens. ( B) Astrocytes were transfected as in A for 48 h prior to incubating with control or ER stressed HT-22 conditioned media (UT or Thaps NCM) for 6 h. IL-6, CCL2 and CCL20 mRNA expression was quantified using RT-qPCR. N = 3 independent cell culture preparations, **P ≤ 0.01, *** P ≤ 0.001 determined by two-way ANOVA with Bonferroni’s multiple comparisons test.

    Article Snippet: Thapsigargin (Thaps; 586005–1MG) and tunicamycin (65–438-010MG) were from Calbiochem.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Cell Culture

    Conditioned media from ER stressed astrocytes confer stress resistance to neurons. (A) Murine HT-22 hippocampal neurons were preconditioned with 25% control (UTA or UT ACM) or ER stressed ACM (TA or Thaps ACM) for 6 h, followed by a 24 h recovery period with fresh media. Cells were then treated with Thaps for 4 h. GRP78, phosphorylated JNK (P-JNK) and CHOP protein expression was assessed using immunoblot analysis. (B) CHOP proteins levels were quantified and normalized to GAPDH. N = 3 independent cell culture preparations, *P ≤ 0.05 determined by two-way ANOVA with Tukey’s multiple comparisons test. ( C ) Caspase-3 activity was analyzed by measuring the fluorescence intensity of the cleaved caspase-3 substrate Ac-DEVD-AMC after preconditioned HT-22 neurons were stimulated with either the vehicle (DMSO) or 0.1 μM thapsigargin (Thaps) for 4 h or ( D ) 18 h. N = 3, *P ≤ 0.05, **P ≤ 0.01 determined by two-way ANOVA with Tukey’s multiple comparisons test.

    Journal: Journal of neurochemistry

    Article Title: Endoplasmic reticulum stress is transmissible in vitro between cells of the central nervous system

    doi: 10.1111/jnc.14642

    Figure Lengend Snippet: Conditioned media from ER stressed astrocytes confer stress resistance to neurons. (A) Murine HT-22 hippocampal neurons were preconditioned with 25% control (UTA or UT ACM) or ER stressed ACM (TA or Thaps ACM) for 6 h, followed by a 24 h recovery period with fresh media. Cells were then treated with Thaps for 4 h. GRP78, phosphorylated JNK (P-JNK) and CHOP protein expression was assessed using immunoblot analysis. (B) CHOP proteins levels were quantified and normalized to GAPDH. N = 3 independent cell culture preparations, *P ≤ 0.05 determined by two-way ANOVA with Tukey’s multiple comparisons test. ( C ) Caspase-3 activity was analyzed by measuring the fluorescence intensity of the cleaved caspase-3 substrate Ac-DEVD-AMC after preconditioned HT-22 neurons were stimulated with either the vehicle (DMSO) or 0.1 μM thapsigargin (Thaps) for 4 h or ( D ) 18 h. N = 3, *P ≤ 0.05, **P ≤ 0.01 determined by two-way ANOVA with Tukey’s multiple comparisons test.

    Article Snippet: Thapsigargin (Thaps; 586005–1MG) and tunicamycin (65–438-010MG) were from Calbiochem.

    Techniques: Expressing, Cell Culture, Activity Assay, Fluorescence