caspase reaction buffer  (Millipore)


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    Structured Review

    Millipore caspase reaction buffer
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Caspase Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase reaction buffer/product/Millipore
    Average 82 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    caspase reaction buffer - by Bioz Stars, 2020-02
    82/100 stars

    Images

    1) Product Images from "Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *"

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M800237200

    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Figure Legend Snippet: TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Techniques Used: Activation Assay

    2) Product Images from "Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells"

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Akt1 AS sensitized cancer cells to chemotherapeutic drugs. At day 0, 20,000 of H460 or NHF, or 40,000 of HCT15 cells (per well) were plated into 96-well Costar plates. At day 1, cells were transfected with 250 nM Akt1 AS, or MS, or lipofectin control as described in Materials and Methods section. After 24 hours, 1.5 µM doxorubicin was added to HCT15 cells for 15 hours (B), to H460 cells for 8 hours (A), and to NHF cells for 24 hours (C) followed by caspase activity assay as described in Materials and Methods. In another experiment, 24 hours after transfection, 100 µM etoposide was added to H460 (D), or HCT-15 (E), or NHF cells (F) for 24 hours followed by caspase activity assay as described in Materials and Methods. Data are representative of two independent experiments with four replicates for each point. Shown is the mean±SD of the four replicates.
    Figure Legend Snippet: Akt1 AS sensitized cancer cells to chemotherapeutic drugs. At day 0, 20,000 of H460 or NHF, or 40,000 of HCT15 cells (per well) were plated into 96-well Costar plates. At day 1, cells were transfected with 250 nM Akt1 AS, or MS, or lipofectin control as described in Materials and Methods section. After 24 hours, 1.5 µM doxorubicin was added to HCT15 cells for 15 hours (B), to H460 cells for 8 hours (A), and to NHF cells for 24 hours (C) followed by caspase activity assay as described in Materials and Methods. In another experiment, 24 hours after transfection, 100 µM etoposide was added to H460 (D), or HCT-15 (E), or NHF cells (F) for 24 hours followed by caspase activity assay as described in Materials and Methods. Data are representative of two independent experiments with four replicates for each point. Shown is the mean±SD of the four replicates.

    Techniques Used: Transfection, Mass Spectrometry, Caspase Activity Assay

    Related Articles

    Transfection:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: SREBP-1 expression was then restored by transfection with a pcDNA3.1 plasmid containing a cDNA for the expression of the caspase site mutants SREBP-1(D460A) for the human HepG2 cells and SREBP-1(D451A) for the mouse McA-RH 7777 cells, respectively. .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    In Vitro:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: Purified SREBP-1 containing a histidine tag was generated by in vitro transcription and translation followed by purification on a nickel resin. .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Fluorescence:

    Article Title: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells
    Article Snippet: Different amounts of caspase proteins (100 μg for caspase 3 and 200 μg for caspase 9) were diluted in 250 μl reporter lysis buffer and incubated with equal volumes of 2x caspase reaction buffer containing 100 μM fluorogenic substrates for caspase 3 or 9 (Calbiochem) at 37 °C for 3 hours. .. The intensity of emitted fluorescence at 505 nm was measured using a fluorescence spectrometer (Bio-Tek).

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells
    Article Snippet: Cells with 100 µ l culture medium in 96-well plate were lysed in 20 µ l of lysis buffer (in mM: 10 Hepes, pH 7.5, 35 KCl, 4 MgCl2 , 0.1 EDTA, 0.1 EGTA, 0.2 PMSF, 1.0 DTT, 1xprotease inhibitor cocktail tablet; Boehringer Mannheim, Mannheim, Germany) at room temperature for 20 minutes, followed by addition of 80 µ l of the caspase reaction buffer containing 48 mM Hepes, pH 7.5, 292 mM sucrose, 0.1% CHAPS (Calbiochem, San Diego, CA), 1 mM Ac-DEVD-AMC (Bachem, King of Prussia, PA). .. The units of fluorescence change per hour (dFU/h) are defined as caspase activity.

    Generated:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: Purified SREBP-1 containing a histidine tag was generated by in vitro transcription and translation followed by purification on a nickel resin. .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Purification:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol). .. TNF α Provokes a Cholesterol-independent Activation of SREBP-1 in Ethanol-exposed Cells —Exposure of the rat hepatoma cell line McA-RH 7777 to ethanol has been shown to activate SREBP-1 ( ).

    Incubation:

    Article Title: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells
    Article Snippet: .. Different amounts of caspase proteins (100 μg for caspase 3 and 200 μg for caspase 9) were diluted in 250 μl reporter lysis buffer and incubated with equal volumes of 2x caspase reaction buffer containing 100 μM fluorogenic substrates for caspase 3 or 9 (Calbiochem) at 37 °C for 3 hours. .. The intensity of emitted fluorescence at 505 nm was measured using a fluorescence spectrometer (Bio-Tek).

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol). .. TNF α Provokes a Cholesterol-independent Activation of SREBP-1 in Ethanol-exposed Cells —Exposure of the rat hepatoma cell line McA-RH 7777 to ethanol has been shown to activate SREBP-1 ( ).

    Activity Assay:

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells
    Article Snippet: Cells with 100 µ l culture medium in 96-well plate were lysed in 20 µ l of lysis buffer (in mM: 10 Hepes, pH 7.5, 35 KCl, 4 MgCl2 , 0.1 EDTA, 0.1 EGTA, 0.2 PMSF, 1.0 DTT, 1xprotease inhibitor cocktail tablet; Boehringer Mannheim, Mannheim, Germany) at room temperature for 20 minutes, followed by addition of 80 µ l of the caspase reaction buffer containing 48 mM Hepes, pH 7.5, 292 mM sucrose, 0.1% CHAPS (Calbiochem, San Diego, CA), 1 mM Ac-DEVD-AMC (Bachem, King of Prussia, PA). .. The units of fluorescence change per hour (dFU/h) are defined as caspase activity.

    Caspase Activity Assay:

    Article Title: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells
    Article Snippet: Paragraph title: Caspase activity assay ... Different amounts of caspase proteins (100 μg for caspase 3 and 200 μg for caspase 9) were diluted in 250 μl reporter lysis buffer and incubated with equal volumes of 2x caspase reaction buffer containing 100 μM fluorogenic substrates for caspase 3 or 9 (Calbiochem) at 37 °C for 3 hours.

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells
    Article Snippet: Paragraph title: Caspase Activity Assay ... Cells with 100 µ l culture medium in 96-well plate were lysed in 20 µ l of lysis buffer (in mM: 10 Hepes, pH 7.5, 35 KCl, 4 MgCl2 , 0.1 EDTA, 0.1 EGTA, 0.2 PMSF, 1.0 DTT, 1xprotease inhibitor cocktail tablet; Boehringer Mannheim, Mannheim, Germany) at room temperature for 20 minutes, followed by addition of 80 µ l of the caspase reaction buffer containing 48 mM Hepes, pH 7.5, 292 mM sucrose, 0.1% CHAPS (Calbiochem, San Diego, CA), 1 mM Ac-DEVD-AMC (Bachem, King of Prussia, PA).

    Expressing:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: SREBP-1 expression was then restored by transfection with a pcDNA3.1 plasmid containing a cDNA for the expression of the caspase site mutants SREBP-1(D460A) for the human HepG2 cells and SREBP-1(D451A) for the mouse McA-RH 7777 cells, respectively. .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Lysis:

    Article Title: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells
    Article Snippet: .. Different amounts of caspase proteins (100 μg for caspase 3 and 200 μg for caspase 9) were diluted in 250 μl reporter lysis buffer and incubated with equal volumes of 2x caspase reaction buffer containing 100 μM fluorogenic substrates for caspase 3 or 9 (Calbiochem) at 37 °C for 3 hours. .. The intensity of emitted fluorescence at 505 nm was measured using a fluorescence spectrometer (Bio-Tek).

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells
    Article Snippet: .. Cells with 100 µ l culture medium in 96-well plate were lysed in 20 µ l of lysis buffer (in mM: 10 Hepes, pH 7.5, 35 KCl, 4 MgCl2 , 0.1 EDTA, 0.1 EGTA, 0.2 PMSF, 1.0 DTT, 1xprotease inhibitor cocktail tablet; Boehringer Mannheim, Mannheim, Germany) at room temperature for 20 minutes, followed by addition of 80 µ l of the caspase reaction buffer containing 48 mM Hepes, pH 7.5, 292 mM sucrose, 0.1% CHAPS (Calbiochem, San Diego, CA), 1 mM Ac-DEVD-AMC (Bachem, King of Prussia, PA). .. After mixing, the plate was read on Cytofluor series 4000 (Applied Biosystems) using the following setting: excitation=360/40, emission=460/ 40, gain=38, cycle=1.

    Recombinant:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol). .. TNF α Provokes a Cholesterol-independent Activation of SREBP-1 in Ethanol-exposed Cells —Exposure of the rat hepatoma cell line McA-RH 7777 to ethanol has been shown to activate SREBP-1 ( ).

    Plasmid Preparation:

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *
    Article Snippet: SREBP-1 expression was then restored by transfection with a pcDNA3.1 plasmid containing a cDNA for the expression of the caspase site mutants SREBP-1(D460A) for the human HepG2 cells and SREBP-1(D451A) for the mouse McA-RH 7777 cells, respectively. .. The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

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    Millipore caspase reaction buffer
    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by <t>caspase-4</t> or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.
    Caspase Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase reaction buffer/product/Millipore
    Average 82 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    caspase reaction buffer - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    94
    Millipore caspase assay buffer
    ONC201 provokes apoptosis in human lung cancer cells. A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative <t>caspase-8/-3</t> activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. * p
    Caspase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase assay buffer/product/Millipore
    Average 94 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    caspase assay buffer - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    78
    Millipore pp2a specific reaction buffer
    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, <t>PP2A-C</t> and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).
    Pp2a Specific Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pp2a specific reaction buffer/product/Millipore
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pp2a specific reaction buffer - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    Image Search Results


    TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor-? Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive *

    doi: 10.1074/jbc.M800237200

    Figure Lengend Snippet: TNF provokes a cholesterol-insensitive processing of SREBP-1 in ethanol-exposed cells that is promoted by caspase-4 or -12 activation, resulting in the inappropriate induction of lipogenic enzymes and consequent accumulation of triglycerides. DISC , death inducing signaling complex.

    Article Snippet: The purified SREBP-1 was incubated with 5 units of recombinant caspase-3, -4, or -2 (EMD Biosciences) in caspase reaction buffer (50 m m HEPES, pH 7.2, 50 m m NaCl, 0.1% CHAPS, 10 m m EDTA, 5% glycerol, and 10 m m dithiothreitol).

    Techniques: Activation Assay

    Akt1 AS sensitized cancer cells to chemotherapeutic drugs. At day 0, 20,000 of H460 or NHF, or 40,000 of HCT15 cells (per well) were plated into 96-well Costar plates. At day 1, cells were transfected with 250 nM Akt1 AS, or MS, or lipofectin control as described in Materials and Methods section. After 24 hours, 1.5 µM doxorubicin was added to HCT15 cells for 15 hours (B), to H460 cells for 8 hours (A), and to NHF cells for 24 hours (C) followed by caspase activity assay as described in Materials and Methods. In another experiment, 24 hours after transfection, 100 µM etoposide was added to H460 (D), or HCT-15 (E), or NHF cells (F) for 24 hours followed by caspase activity assay as described in Materials and Methods. Data are representative of two independent experiments with four replicates for each point. Shown is the mean±SD of the four replicates.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells

    doi:

    Figure Lengend Snippet: Akt1 AS sensitized cancer cells to chemotherapeutic drugs. At day 0, 20,000 of H460 or NHF, or 40,000 of HCT15 cells (per well) were plated into 96-well Costar plates. At day 1, cells were transfected with 250 nM Akt1 AS, or MS, or lipofectin control as described in Materials and Methods section. After 24 hours, 1.5 µM doxorubicin was added to HCT15 cells for 15 hours (B), to H460 cells for 8 hours (A), and to NHF cells for 24 hours (C) followed by caspase activity assay as described in Materials and Methods. In another experiment, 24 hours after transfection, 100 µM etoposide was added to H460 (D), or HCT-15 (E), or NHF cells (F) for 24 hours followed by caspase activity assay as described in Materials and Methods. Data are representative of two independent experiments with four replicates for each point. Shown is the mean±SD of the four replicates.

    Article Snippet: Cells with 100 µ l culture medium in 96-well plate were lysed in 20 µ l of lysis buffer (in mM: 10 Hepes, pH 7.5, 35 KCl, 4 MgCl2 , 0.1 EDTA, 0.1 EGTA, 0.2 PMSF, 1.0 DTT, 1xprotease inhibitor cocktail tablet; Boehringer Mannheim, Mannheim, Germany) at room temperature for 20 minutes, followed by addition of 80 µ l of the caspase reaction buffer containing 48 mM Hepes, pH 7.5, 292 mM sucrose, 0.1% CHAPS (Calbiochem, San Diego, CA), 1 mM Ac-DEVD-AMC (Bachem, King of Prussia, PA).

    Techniques: Transfection, Mass Spectrometry, Caspase Activity Assay

    ONC201 provokes apoptosis in human lung cancer cells. A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative caspase-8/-3 activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. * p

    Journal: PLoS ONE

    Article Title: Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study

    doi: 10.1371/journal.pone.0162133

    Figure Lengend Snippet: ONC201 provokes apoptosis in human lung cancer cells. A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative caspase-8/-3 activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. * p

    Article Snippet: Caspase activity assay After treatment of cells, cytosolic proteins (30 μg lysates per treatment) were added to the caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (10 μg/mL, CalBiochem) as the caspase-3 substrate, or Ac-IETD-AFC (10 μg/mL, CalBiochem) as the caspase-8 substrate.

    Techniques: Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, FACS

    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Incubation, Immunoprecipitation, Activity Assay, Phosphatase Assay, Injection, Mouse Assay, Purification, In Vitro

    Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay, Stable Transfection, Negative Control, Activity Assay, Incubation, Mouse Assay

    Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Binding Assay, Inhibition

    PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Injection, Mouse Assay, Activity Assay, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay