anti nqo1 antibody Cell Signaling Technology Inc Search Results


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  • 99
    Cell Signaling Technology Inc anti nqo1
    Dicoumarol, an <t>NQO1</t> inhibitor, blocks the apoptotic effects of β-lapachone. (A) CL1-1 cells (top) or CL1-5 cells (bottom) were left untreated or were incubated for 6 h with 5 µM β-lapachone and/or 10 µM dicoumarol, then stained with Annexin V-FITC and the Annexin V fluorescence measured by flow cytometry. (B) Morphological changes after drug treatment. CL1-1 or CL1-5 cells were left untreated (CTL) or were incubated for 24 h with 5 µM β-lapachone with or without 10 µM dicoumarol, then stained with acridine orange to observe the morphology of the cell nucleus. The scale bar represents 50 µm.
    Anti Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc mouse anti nqo1
    Polydatin enhances miR-200a expression targeting Keap1 to activate Nrf2 antioxidant pathway in suppression of oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A) qRT-PCR analysis of miR-200a expression levels in BRL-3A (4 h) and HepG2 cells (12 h) (n = 4 at least). (B) Diagrams showed the miR-200a putative binding sites and corresponding mutant sites of Keap1. Dual-luciferase reporter assay of miR-200a with 3′UTR vectors (wild type or mutant) of rat keap1 in BRL-3A and HepG2 cells (n = 4 at least). (C) qRT-PCR analysis of miR-200a expression in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (4 h) (n = 4 at least). (D) Western blot analysis of Keap1, nuclear Nrf2, GST, HO-1 and <t>NQO1</t> protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 8) in 50 nM miR-200a mimic transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative miR-200a levels were normalized to U6. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
    Mouse Anti Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cell Signaling Technology Inc nqo1 a180 mouse mab
    Polydatin enhances miR-200a expression targeting Keap1 to activate Nrf2 antioxidant pathway in suppression of oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A) qRT-PCR analysis of miR-200a expression levels in BRL-3A (4 h) and HepG2 cells (12 h) (n = 4 at least). (B) Diagrams showed the miR-200a putative binding sites and corresponding mutant sites of Keap1. Dual-luciferase reporter assay of miR-200a with 3′UTR vectors (wild type or mutant) of rat keap1 in BRL-3A and HepG2 cells (n = 4 at least). (C) qRT-PCR analysis of miR-200a expression in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (4 h) (n = 4 at least). (D) Western blot analysis of Keap1, nuclear Nrf2, GST, HO-1 and <t>NQO1</t> protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 8) in 50 nM miR-200a mimic transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative miR-200a levels were normalized to U6. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P
    Nqo1 A180 Mouse Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti nqo1 antibody
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Rabbit Anti Nqo1 Antibody, supplied by Cell Signaling Technology Inc, 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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    92
    Cell Signaling Technology Inc mouse anti nad
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Mouse Anti Nad, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc nqo1 d6h3a rabbit mab
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Nqo1 D6h3a Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc anti nqo 1 antibody
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Anti Nqo 1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc anti nqo1 3187 antibodies
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Anti Nqo1 3187 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti nqo1 a180
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Anti Nqo1 A180, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti nqo1 rabbit monoclonal antibody
    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, <t>NQO1,</t> and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p
    Anti Nqo1 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc nadph quinone oxidoreductase
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Nadph Quinone Oxidoreductase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc mouse anti nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Mouse Anti Nicotinamide Adenine Dinucleotide Phosphate Dehydrogenase Quinone 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse polyclonal anti nqo1 antibody
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Mouse Polyclonal Anti Nqo1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc primary monoclonal antibodies
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Primary Monoclonal Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dicoumarol, an NQO1 inhibitor, blocks the apoptotic effects of β-lapachone. (A) CL1-1 cells (top) or CL1-5 cells (bottom) were left untreated or were incubated for 6 h with 5 µM β-lapachone and/or 10 µM dicoumarol, then stained with Annexin V-FITC and the Annexin V fluorescence measured by flow cytometry. (B) Morphological changes after drug treatment. CL1-1 or CL1-5 cells were left untreated (CTL) or were incubated for 24 h with 5 µM β-lapachone with or without 10 µM dicoumarol, then stained with acridine orange to observe the morphology of the cell nucleus. The scale bar represents 50 µm.

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: Dicoumarol, an NQO1 inhibitor, blocks the apoptotic effects of β-lapachone. (A) CL1-1 cells (top) or CL1-5 cells (bottom) were left untreated or were incubated for 6 h with 5 µM β-lapachone and/or 10 µM dicoumarol, then stained with Annexin V-FITC and the Annexin V fluorescence measured by flow cytometry. (B) Morphological changes after drug treatment. CL1-1 or CL1-5 cells were left untreated (CTL) or were incubated for 24 h with 5 µM β-lapachone with or without 10 µM dicoumarol, then stained with acridine orange to observe the morphology of the cell nucleus. The scale bar represents 50 µm.

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Incubation, Staining, Fluorescence, Flow Cytometry, Cytometry, CTL Assay

    β-lapachone-induced cell death is associated with NQO1 expression levels. (A) Percentage survival of the lung cancer cell lines CL1-1, CL1-5, and A549. Cells were treated with 0–10 µM β-lapachone for 12 h, then cell viability was determined by crystal violet staining assay and expressed as a percentage of the value for cultures with no β-lapachone. (B–D) NQO1 activity levels (B), NQO1 RNA expression levels (C), and NQO1 protein expression levels (D) in the three lung cancer cell lines grown under normal culture conditions. (E) Percentage survival of A549 cells (left panel), CL1-1 cells (center panel), and CL1-5 cells (right panel) incubated with the indicated concentration of β-lapachone for 3, 6, 12, or 24 h examined by crystal violet staining and expressed as percentage survival compared to the untreated cells. The results are the mean ± SD for 3 independent experiments, each in triplicate.

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: β-lapachone-induced cell death is associated with NQO1 expression levels. (A) Percentage survival of the lung cancer cell lines CL1-1, CL1-5, and A549. Cells were treated with 0–10 µM β-lapachone for 12 h, then cell viability was determined by crystal violet staining assay and expressed as a percentage of the value for cultures with no β-lapachone. (B–D) NQO1 activity levels (B), NQO1 RNA expression levels (C), and NQO1 protein expression levels (D) in the three lung cancer cell lines grown under normal culture conditions. (E) Percentage survival of A549 cells (left panel), CL1-1 cells (center panel), and CL1-5 cells (right panel) incubated with the indicated concentration of β-lapachone for 3, 6, 12, or 24 h examined by crystal violet staining and expressed as percentage survival compared to the untreated cells. The results are the mean ± SD for 3 independent experiments, each in triplicate.

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Expressing, Staining, Activity Assay, RNA Expression, Incubation, Concentration Assay

    NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on β-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (−) or NQO1 siRNA (+) for 24 h, then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C), then 2 µM β-lapachone or medium was added and the cells incubated for 12 h, when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on β-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (−) or NQO1 siRNA (+) for 24 h, then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C), then 2 µM β-lapachone or medium was added and the cells incubated for 12 h, when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Transfection, Incubation, Staining

    The knockdown effects of NQO1 siRNA on NQO1 RNA, protein, and activity. (A–C) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1, then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac, sulindac sulfide, or sulindac sulfone for 6 or 24 h, then NQO1 activity was measured. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: The knockdown effects of NQO1 siRNA on NQO1 RNA, protein, and activity. (A–C) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1, then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac, sulindac sulfide, or sulindac sulfone for 6 or 24 h, then NQO1 activity was measured. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Activity Assay, Transfection, CTL Assay, RNA Expression, Polymerase Chain Reaction, Expressing, Western Blot, Incubation

    The increase in β-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor, dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C) with or without 10 µM dicoumarol, then were incubated for a further 12 h with or without addition of 2 µM β-lapachone, then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: The increase in β-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor, dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C) with or without 10 µM dicoumarol, then were incubated for a further 12 h with or without addition of 2 µM β-lapachone, then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Incubation, Staining

    Sulindac and its metabolites increase NQO1 expression and activity. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were incubated with 100 or 250 µM sulindac, sulindac sulfone, or sulindac sulfide for 6, 12, or 24 h, then protein levels were measured by Western blotting. (B–D) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (Ctl) or were incubated with the indicated concentration of sulindac (B), sulindac sulfone (C), or sulindac sulfide (D) for the indicated time, then NQO1 activity was measured. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: Sulindac and its metabolites increase NQO1 expression and activity. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were incubated with 100 or 250 µM sulindac, sulindac sulfone, or sulindac sulfide for 6, 12, or 24 h, then protein levels were measured by Western blotting. (B–D) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (Ctl) or were incubated with the indicated concentration of sulindac (B), sulindac sulfone (C), or sulindac sulfide (D) for the indicated time, then NQO1 activity was measured. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Expressing, Activity Assay, Incubation, Western Blot, CTL Assay, Concentration Assay

    The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Journal: PLoS ONE

    Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

    doi: 10.1371/journal.pone.0009631

    Figure Lengend Snippet: The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Article Snippet: Analysis of Nrf2 Activation Western blot analysis of nuclear protein fractions was performed by Odyssey system (LI-COR Bioscience, Nebraska USA), using antibodies specific for Nrf2, TBP, beta-actin (Santa Cruz Technology), NQO1 (Cell Signaling Technology).

    Techniques: Mouse Assay, Injection, Staining, MANN-WHITNEY, Two Tailed Test, Activation Assay, Western Blot

    Expression of Nrf2 downstreams and Akt is effected by the expression of LMP1 with mutation CTAR1 regions. 293T cells were transfected with wild-type LMP1(WT) or Ctar1 mutant of LMP1 (ΔCtar1) and empty vector. Cell lysates were harvested 28 h after transfection, and western blot were performed with pAkt, Akt, NQO-1, HO-1 and b-actin antibody. b-actin was used as equal loading controls for normalization. The fold increase in Nrf2 and HO-1 is indicated numerically, as determined by densitometry.

    Journal: Translational Oncology

    Article Title: LMP1 and 2A Induce the Expression of Nrf2 Through Akt Signaling Pathway in Epstein-Barr Virus–Transformed B Cells

    doi: 10.1016/j.tranon.2019.02.009

    Figure Lengend Snippet: Expression of Nrf2 downstreams and Akt is effected by the expression of LMP1 with mutation CTAR1 regions. 293T cells were transfected with wild-type LMP1(WT) or Ctar1 mutant of LMP1 (ΔCtar1) and empty vector. Cell lysates were harvested 28 h after transfection, and western blot were performed with pAkt, Akt, NQO-1, HO-1 and b-actin antibody. b-actin was used as equal loading controls for normalization. The fold increase in Nrf2 and HO-1 is indicated numerically, as determined by densitometry.

    Article Snippet: We used anti–β-actin, anti–Lamin B and LMP2A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LMP1 (Abcam, Cambridge, MA), anti-Nrf2, anti-Ho-1, anti–NQO-1, anti-pAkt, anti-Akt, anti–caspase-9, and anti–caspase-3 (Cell Signaling Technology, Beverly, MA) antibodies.

    Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Western Blot

    Suppression of Nrf2 induces apoptotic cell death in EBV-transformed B cells. EBV-transformed B cells were transfected with siRNA against Nrf2 for 48 hours. (A) Cell extracts were prepared from siRNA-Nrf2–transfected EBV-transformed B cells. The protein levels of Nrf2, HO-1, and NQO-1 were measured by Western blot analysis. The fold increase in Nrf2, NQO-1, and HO-1 is indicated numerically, as determined by densitometry. (B) Cell viability was evaluated by the MTX assay. The data are expressed as the mean ± S.D. * P

    Journal: Translational Oncology

    Article Title: LMP1 and 2A Induce the Expression of Nrf2 Through Akt Signaling Pathway in Epstein-Barr Virus–Transformed B Cells

    doi: 10.1016/j.tranon.2019.02.009

    Figure Lengend Snippet: Suppression of Nrf2 induces apoptotic cell death in EBV-transformed B cells. EBV-transformed B cells were transfected with siRNA against Nrf2 for 48 hours. (A) Cell extracts were prepared from siRNA-Nrf2–transfected EBV-transformed B cells. The protein levels of Nrf2, HO-1, and NQO-1 were measured by Western blot analysis. The fold increase in Nrf2, NQO-1, and HO-1 is indicated numerically, as determined by densitometry. (B) Cell viability was evaluated by the MTX assay. The data are expressed as the mean ± S.D. * P

    Article Snippet: We used anti–β-actin, anti–Lamin B and LMP2A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LMP1 (Abcam, Cambridge, MA), anti-Nrf2, anti-Ho-1, anti–NQO-1, anti-pAkt, anti-Akt, anti–caspase-9, and anti–caspase-3 (Cell Signaling Technology, Beverly, MA) antibodies.

    Techniques: Transformation Assay, Transfection, Western Blot

    Level of mRNA expression of Nrf2, HO-1 and NQO-1. Graphs for expression level of Nrf2, NQO-1 and HO-1 were quantified using the ratio against b-actin from (A) PBMCs and EBV-transformed B cells or (B) LMP1 or LMP2A or (D) Nrf2-transfected or (C) LY294002 treated cells. The mRNA levels of Nrf2, HO-1, and NQO-1 were measured by Real-time PCR. The data are expressed as the mean ± S.D. * p

    Journal: Translational Oncology

    Article Title: LMP1 and 2A Induce the Expression of Nrf2 Through Akt Signaling Pathway in Epstein-Barr Virus–Transformed B Cells

    doi: 10.1016/j.tranon.2019.02.009

    Figure Lengend Snippet: Level of mRNA expression of Nrf2, HO-1 and NQO-1. Graphs for expression level of Nrf2, NQO-1 and HO-1 were quantified using the ratio against b-actin from (A) PBMCs and EBV-transformed B cells or (B) LMP1 or LMP2A or (D) Nrf2-transfected or (C) LY294002 treated cells. The mRNA levels of Nrf2, HO-1, and NQO-1 were measured by Real-time PCR. The data are expressed as the mean ± S.D. * p

    Article Snippet: We used anti–β-actin, anti–Lamin B and LMP2A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LMP1 (Abcam, Cambridge, MA), anti-Nrf2, anti-Ho-1, anti–NQO-1, anti-pAkt, anti-Akt, anti–caspase-9, and anti–caspase-3 (Cell Signaling Technology, Beverly, MA) antibodies.

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

    Nrf2 activation is upregulated in EBV-infected B cells. (A) Whole cell extract and (B) cytosolic and nuclear extracts were prepared from PBMCs and EBV-transformed B cells. The protein levels of Nrf2, HO-1, and NQO-1 were measured by Western blot analysis. β-Actin and Lamin B1 were used as equal loading control for normalization. CE , cytosol extract; NE , nuclear extract. The fold increase in Nrf2 and HO-1 is indicated numerically, as determined by densitometry. (C) The expression level of Nrf2 was detected by immunofluorescence. DAPI was used to counter stain the nucleus. Photographs were taken at 200× magnification. (D) EBV-transformed B cells were transfected with siRNA against Nrf2 for 48 hours. ROS were detected with carboxy-H 2 DCFDA. The data are expressed as the mean ± S.D. * P

    Journal: Translational Oncology

    Article Title: LMP1 and 2A Induce the Expression of Nrf2 Through Akt Signaling Pathway in Epstein-Barr Virus–Transformed B Cells

    doi: 10.1016/j.tranon.2019.02.009

    Figure Lengend Snippet: Nrf2 activation is upregulated in EBV-infected B cells. (A) Whole cell extract and (B) cytosolic and nuclear extracts were prepared from PBMCs and EBV-transformed B cells. The protein levels of Nrf2, HO-1, and NQO-1 were measured by Western blot analysis. β-Actin and Lamin B1 were used as equal loading control for normalization. CE , cytosol extract; NE , nuclear extract. The fold increase in Nrf2 and HO-1 is indicated numerically, as determined by densitometry. (C) The expression level of Nrf2 was detected by immunofluorescence. DAPI was used to counter stain the nucleus. Photographs were taken at 200× magnification. (D) EBV-transformed B cells were transfected with siRNA against Nrf2 for 48 hours. ROS were detected with carboxy-H 2 DCFDA. The data are expressed as the mean ± S.D. * P

    Article Snippet: We used anti–β-actin, anti–Lamin B and LMP2A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LMP1 (Abcam, Cambridge, MA), anti-Nrf2, anti-Ho-1, anti–NQO-1, anti-pAkt, anti-Akt, anti–caspase-9, and anti–caspase-3 (Cell Signaling Technology, Beverly, MA) antibodies.

    Techniques: Activation Assay, Infection, Transformation Assay, Western Blot, Expressing, Immunofluorescence, Staining, Transfection

    Akt activates Nrf2 in EBV-transformed B cells. (A) Cell extracts were prepared from PBMCs and EBV-transformed B cells. The protein levels of pAkt and Akt were measured by Western blot analysis. (B-D) EBV-transformed B cells were treated with LY294002 (10 μM) for 48 hours. (B and D) Whole cell extract and (C) cytosolic and nuclear extracts were prepared from LY294002-treated cells. The protein levels of Nrf2, pAkt, Akt, HO-1, and NQO-1 were measured by Western blot analysis. β-Actin and Lamin B1 were used as equal loading controls for normalization. CE , cytosol extract; NE , nuclear extract. (E) Cell extracts were prepared from siRNA-LMP1 or LMP2A-transfected EBV-transformed B cells. The protein levels of pAkt and Akt were measured by Western blot analysis. The fold increase in pAkt, Nrf2, NQO-1, and HO-1 is indicated numerically, as determined by densitometry.

    Journal: Translational Oncology

    Article Title: LMP1 and 2A Induce the Expression of Nrf2 Through Akt Signaling Pathway in Epstein-Barr Virus–Transformed B Cells

    doi: 10.1016/j.tranon.2019.02.009

    Figure Lengend Snippet: Akt activates Nrf2 in EBV-transformed B cells. (A) Cell extracts were prepared from PBMCs and EBV-transformed B cells. The protein levels of pAkt and Akt were measured by Western blot analysis. (B-D) EBV-transformed B cells were treated with LY294002 (10 μM) for 48 hours. (B and D) Whole cell extract and (C) cytosolic and nuclear extracts were prepared from LY294002-treated cells. The protein levels of Nrf2, pAkt, Akt, HO-1, and NQO-1 were measured by Western blot analysis. β-Actin and Lamin B1 were used as equal loading controls for normalization. CE , cytosol extract; NE , nuclear extract. (E) Cell extracts were prepared from siRNA-LMP1 or LMP2A-transfected EBV-transformed B cells. The protein levels of pAkt and Akt were measured by Western blot analysis. The fold increase in pAkt, Nrf2, NQO-1, and HO-1 is indicated numerically, as determined by densitometry.

    Article Snippet: We used anti–β-actin, anti–Lamin B and LMP2A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LMP1 (Abcam, Cambridge, MA), anti-Nrf2, anti-Ho-1, anti–NQO-1, anti-pAkt, anti-Akt, anti–caspase-9, and anti–caspase-3 (Cell Signaling Technology, Beverly, MA) antibodies.

    Techniques: Transformation Assay, Western Blot, Transfection

    Induction of GST and NQO1 by ITCs in NBT-II cells. Cells were treated with each ITC (see for name and chemical structure) at 0, 3.75 (□) or 7.5 μM (■) for 24 h and then harvested for measurement of GST and NQO1 activities.

    Journal:

    Article Title: Structure-activity relationships and organ specificity in the induction of GST and NQO1 by alkyl-aryl isothiocyanates

    doi: 10.1007/s11095-008-9595-2

    Figure Lengend Snippet: Induction of GST and NQO1 by ITCs in NBT-II cells. Cells were treated with each ITC (see for name and chemical structure) at 0, 3.75 (□) or 7.5 μM (■) for 24 h and then harvested for measurement of GST and NQO1 activities.

    Article Snippet: The anti-NQO1 antibody was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques:

    Induction of GST and NQO1 by ITCs in rat organs. Groups of 6 rats were dosed with each ITC (see for name and chemical structure) in soya oil at a dose level of 250 μmoles/kg/day for 5 days. A further group of animals received soya oil alone.

    Journal:

    Article Title: Structure-activity relationships and organ specificity in the induction of GST and NQO1 by alkyl-aryl isothiocyanates

    doi: 10.1007/s11095-008-9595-2

    Figure Lengend Snippet: Induction of GST and NQO1 by ITCs in rat organs. Groups of 6 rats were dosed with each ITC (see for name and chemical structure) in soya oil at a dose level of 250 μmoles/kg/day for 5 days. A further group of animals received soya oil alone.

    Article Snippet: The anti-NQO1 antibody was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques:

    Effect of benzyl ITC on expression of GST and NQO1. NBT-II cells were treated with benzyl ITC at the specified concentrations for 24 h and then harvested for measurement of expression of GST-mu and NQO1 by Western blot analysis. GAPDH was used as a loading

    Journal:

    Article Title: Structure-activity relationships and organ specificity in the induction of GST and NQO1 by alkyl-aryl isothiocyanates

    doi: 10.1007/s11095-008-9595-2

    Figure Lengend Snippet: Effect of benzyl ITC on expression of GST and NQO1. NBT-II cells were treated with benzyl ITC at the specified concentrations for 24 h and then harvested for measurement of expression of GST-mu and NQO1 by Western blot analysis. GAPDH was used as a loading

    Article Snippet: The anti-NQO1 antibody was purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Western Blot

    The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Journal: Nutrients

    Article Title: Pharmacological Investigation of the Anti-Inflammation and Anti-Oxidation Activities of Diallyl Disulfide in a Rat Emphysema Model Induced by Cigarette Smoke Extract

    doi: 10.3390/nu10010079

    Figure Lengend Snippet: The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Article Snippet: Anti-NF-κB p65, i-κB, and NQO1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    NQO1 controls mitotic progression. (A) Control (ctr) siRNA and NQO1 siRNA transfected MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) followed by release in normal medium. Flow cytometric analysis of cell cycle at the indicated time points after release was done using propidium iodide DNA staining. (B) Table shows percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) at indicated time points after nocodazole release from the experiment presented in (A). (C) Control shRNA and NQO1 shRNA stable MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h). Cell cycle distribution was assessed as in A. (D) Mitotic progression of MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA was monitored by live-cell microscopy. Characteristic phase-contrast images are shown. Mitosis is completed in less than 90 min under normal conditions (arrow, upper). Mitosis fails to be completed as evidenced by the lack of formation of two daughter cells (arrow, lower). (E) MCF-7 cells stably expressing H2B-GFP were transfected with either control (ctr) siRNA or NQO1 siRNA. Mitosis was monitored by live-cell microscopy. Characteristic images are shown. Normal completion of mitosis is shown in the upper panel, whereas impaired mitoses are shown in the lower panel. Scale bar is 10 µm. (F) Percentage of cells exhibiting mitotic defects under different experimental conditions. Mitoses were checked in control (ctr) siRNA, NQO1 siRNA and NQO1 siRNA treated with Nicotinamide mononucleotide (ΝΜΝ, 0.5 mΜ) MCF-7 cells.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 controls mitotic progression. (A) Control (ctr) siRNA and NQO1 siRNA transfected MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) followed by release in normal medium. Flow cytometric analysis of cell cycle at the indicated time points after release was done using propidium iodide DNA staining. (B) Table shows percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) at indicated time points after nocodazole release from the experiment presented in (A). (C) Control shRNA and NQO1 shRNA stable MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h). Cell cycle distribution was assessed as in A. (D) Mitotic progression of MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA was monitored by live-cell microscopy. Characteristic phase-contrast images are shown. Mitosis is completed in less than 90 min under normal conditions (arrow, upper). Mitosis fails to be completed as evidenced by the lack of formation of two daughter cells (arrow, lower). (E) MCF-7 cells stably expressing H2B-GFP were transfected with either control (ctr) siRNA or NQO1 siRNA. Mitosis was monitored by live-cell microscopy. Characteristic images are shown. Normal completion of mitosis is shown in the upper panel, whereas impaired mitoses are shown in the lower panel. Scale bar is 10 µm. (F) Percentage of cells exhibiting mitotic defects under different experimental conditions. Mitoses were checked in control (ctr) siRNA, NQO1 siRNA and NQO1 siRNA treated with Nicotinamide mononucleotide (ΝΜΝ, 0.5 mΜ) MCF-7 cells.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Transfection, Flow Cytometry, Staining, shRNA, Microscopy, Stable Transfection, Expressing

    NQO1 and SIRT2 colocalize to the mitotic spindle during mitosis. (A, B) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (A) and telophase (B) in MCF-7 human breast cancer cells. (C, D) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (C) and telophase (D) in BxPC-3 human pancreatic cancer cells. In all panels above, DNA was stained with DAPI (blue). On the right, graphs represent signal intensity scans along the lines drawn for each panel. Scale bar, 20 µm. (E, F, G) Higher magnification images (60x) under same experimental conditions as shown in A, B.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 and SIRT2 colocalize to the mitotic spindle during mitosis. (A, B) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (A) and telophase (B) in MCF-7 human breast cancer cells. (C, D) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (C) and telophase (D) in BxPC-3 human pancreatic cancer cells. In all panels above, DNA was stained with DAPI (blue). On the right, graphs represent signal intensity scans along the lines drawn for each panel. Scale bar, 20 µm. (E, F, G) Higher magnification images (60x) under same experimental conditions as shown in A, B.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Confocal Microscopy, Staining

    NQO1 positively regulates SIRT2 activity. (A) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), NQO1, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 positively regulates SIRT2 activity. (A) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), NQO1, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Activity Assay, Transfection, Western Blot

    NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag- NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag- NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: shRNA, Western Blot, Negative Control, Transfection, Immunoprecipitation, Software, In Vivo, Ubiquitin Assay, Blocking Assay, Plasmid Preparation

    NQO1 interacts directly with SIRT2. (A) MCF-7 cells were transiently transfected with Flag- NQO1 and HA- SIRT2 . 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using either a Flag (left) or an HA (right) antibody followed by western blotting using antibodies against HA and Flag, respectively. Specificity was confirmed by using species-matched control IgG as a negative control. (B) Cell lysates from MCF-7 cells were subjected to immunoprecipitation (IP) using a SIRT2 antibody followed by western blotting using an anti-NQO1 antibody. * denotes Ig heavy and light chain. (C) Cell lysates from MCF-7 cells as described in (A) either unsynchronized or synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) were subjected to immunoprecipitation using an antibody against HA followed by western blotting using a Flag antibody. Specificity was confirmed by using species-matched control IgG as a negative control. Overexpression of both Flag- NQO1 and HA- SIRT2 is confirmed by western blotting (lower). (D) Reciprocal co-immunoprecipitation of endogenous SIRT2 and NQO1 in either unsynchronized or synchronized in mitosis Hela cells. For cell synchronization, HeLa cells were serum starved for 48 h and then released into regular media for 16 h. Cell lysates were subjected to immunoprecipitation (IP) using either a SIRT2 (left) or an NQO1 (right) antibody followed by western blotting using antibodies against SIRT2 and NQO1. Specificity was confirmed by using species-matched control IgG as a negative control. (E) Either unsynchronized or synchronized in mitosis (syn, as described in C) MCF-7 cells were fractionated into cytoplasmic extracts (cyt) and nuclear extracts (nuc). Levels of both SIRT2 and NQO1 were detected by western blotting. Successful cellular fractionation was confirmed by western blotting using antibodies against markers such as Lamin A/C (nuc) and actin (cyt). (F) Recombinant human SIRT2 and GST-NQO1 were used to check protein-protein interaction in vitro . Coomassie staining shows the molecular weight of the two proteins. GST-NQO1 was immunoprecipitated followed by SIRT2 immunodetection of (upper). In addition, SIRT2 was immunoprecipitated first, followed by immunodetection of GST-NQO1 (lower). Beads alone were used in the pull-down assays as negative controls.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 interacts directly with SIRT2. (A) MCF-7 cells were transiently transfected with Flag- NQO1 and HA- SIRT2 . 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using either a Flag (left) or an HA (right) antibody followed by western blotting using antibodies against HA and Flag, respectively. Specificity was confirmed by using species-matched control IgG as a negative control. (B) Cell lysates from MCF-7 cells were subjected to immunoprecipitation (IP) using a SIRT2 antibody followed by western blotting using an anti-NQO1 antibody. * denotes Ig heavy and light chain. (C) Cell lysates from MCF-7 cells as described in (A) either unsynchronized or synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) were subjected to immunoprecipitation using an antibody against HA followed by western blotting using a Flag antibody. Specificity was confirmed by using species-matched control IgG as a negative control. Overexpression of both Flag- NQO1 and HA- SIRT2 is confirmed by western blotting (lower). (D) Reciprocal co-immunoprecipitation of endogenous SIRT2 and NQO1 in either unsynchronized or synchronized in mitosis Hela cells. For cell synchronization, HeLa cells were serum starved for 48 h and then released into regular media for 16 h. Cell lysates were subjected to immunoprecipitation (IP) using either a SIRT2 (left) or an NQO1 (right) antibody followed by western blotting using antibodies against SIRT2 and NQO1. Specificity was confirmed by using species-matched control IgG as a negative control. (E) Either unsynchronized or synchronized in mitosis (syn, as described in C) MCF-7 cells were fractionated into cytoplasmic extracts (cyt) and nuclear extracts (nuc). Levels of both SIRT2 and NQO1 were detected by western blotting. Successful cellular fractionation was confirmed by western blotting using antibodies against markers such as Lamin A/C (nuc) and actin (cyt). (F) Recombinant human SIRT2 and GST-NQO1 were used to check protein-protein interaction in vitro . Coomassie staining shows the molecular weight of the two proteins. GST-NQO1 was immunoprecipitated followed by SIRT2 immunodetection of (upper). In addition, SIRT2 was immunoprecipitated first, followed by immunodetection of GST-NQO1 (lower). Beads alone were used in the pull-down assays as negative controls.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Over Expression, Cell Fractionation, Recombinant, In Vitro, Staining, Molecular Weight, Immunodetection

    Polydatin enhances miR-200a expression targeting Keap1 to activate Nrf2 antioxidant pathway in suppression of oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A) qRT-PCR analysis of miR-200a expression levels in BRL-3A (4 h) and HepG2 cells (12 h) (n = 4 at least). (B) Diagrams showed the miR-200a putative binding sites and corresponding mutant sites of Keap1. Dual-luciferase reporter assay of miR-200a with 3′UTR vectors (wild type or mutant) of rat keap1 in BRL-3A and HepG2 cells (n = 4 at least). (C) qRT-PCR analysis of miR-200a expression in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (4 h) (n = 4 at least). (D) Western blot analysis of Keap1, nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 8) in 50 nM miR-200a mimic transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative miR-200a levels were normalized to U6. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Journal: Redox Biology

    Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

    doi: 10.1016/j.redox.2018.07.002

    Figure Lengend Snippet: Polydatin enhances miR-200a expression targeting Keap1 to activate Nrf2 antioxidant pathway in suppression of oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A) qRT-PCR analysis of miR-200a expression levels in BRL-3A (4 h) and HepG2 cells (12 h) (n = 4 at least). (B) Diagrams showed the miR-200a putative binding sites and corresponding mutant sites of Keap1. Dual-luciferase reporter assay of miR-200a with 3′UTR vectors (wild type or mutant) of rat keap1 in BRL-3A and HepG2 cells (n = 4 at least). (C) qRT-PCR analysis of miR-200a expression in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (4 h) (n = 4 at least). (D) Western blot analysis of Keap1, nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 8) in 50 nM miR-200a mimic transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative miR-200a levels were normalized to U6. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Article Snippet: Rabbit anti-Keap1 (#8047), mouse anti-Lamin A/C (#4777), rabbit anti-GST (#2625), mouse anti-NQO1 (#3187), rabbit anti-NLRP3 (#13158) and HRP-conjugated rabbit anti-IgG (#AP132P) were obtained from Cell Signaling Technology (Cambridge, USA).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Transfection, Incubation, Western Blot

    Polydatin inhibits Keap1 to activate Nrf2 antioxidant pathway and inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) qRT-PCT analysis of Keap1 mRNA levels and Western blot analysis of Keap1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). Relative mRNA levels of Keap1 were normalized to β-actin. (C) Western blot analysis of Keap1 protein levels (24 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. (D) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 6 at least) in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Journal: Redox Biology

    Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

    doi: 10.1016/j.redox.2018.07.002

    Figure Lengend Snippet: Polydatin inhibits Keap1 to activate Nrf2 antioxidant pathway and inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) qRT-PCT analysis of Keap1 mRNA levels and Western blot analysis of Keap1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). Relative mRNA levels of Keap1 were normalized to β-actin. (C) Western blot analysis of Keap1 protein levels (24 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. (D) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (E) assay of ROS levels (24 h) (n = 6 at least) in 50 nM Keap1 siRNA transfected-BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Article Snippet: Rabbit anti-Keap1 (#8047), mouse anti-Lamin A/C (#4777), rabbit anti-GST (#2625), mouse anti-NQO1 (#3187), rabbit anti-NLRP3 (#13158) and HRP-conjugated rabbit anti-IgG (#AP132P) were obtained from Cell Signaling Technology (Cambridge, USA).

    Techniques: Western Blot, Incubation, Transfection

    Polydatin activates Nrf2 antioxidant pathway to inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) Western blot analysis of total and nuclear Nrf2 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (C, D) Western blot analysis of GST, HO-1 and NQO1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (E) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (F) assay of ROS levels (24 h) (n = 5 at laest), (G) Western blot analysis of TXNIP protein levels (48 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. (H) Western blot analysis of nuclear Nrf2 protein levels in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (24 h) (n = 4 at least). Relative protein levels of nuclear Nrf2 were normalized to LaminA, of total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Journal: Redox Biology

    Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

    doi: 10.1016/j.redox.2018.07.002

    Figure Lengend Snippet: Polydatin activates Nrf2 antioxidant pathway to inhibit oxidative stress in fructose-exposed BRL-3A and HepG2 cells. (A, B) Western blot analysis of total and nuclear Nrf2 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (C, D) Western blot analysis of GST, HO-1 and NQO1 protein levels in BRL-3A and HepG2 cells (24 h) (n = 4 at least). (E) Western blot analysis of nuclear Nrf2, GST, HO-1 and NQO1 protein levels (24 h) (n = 4 at least), (F) assay of ROS levels (24 h) (n = 5 at laest), (G) Western blot analysis of TXNIP protein levels (48 h) (n = 4 at least) in 10 μM tBHQ pretreated-BRL-3A cells for 8 h incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone, respectively. (H) Western blot analysis of nuclear Nrf2 protein levels in TXNIP siRNA-transfected BRL-3A cells incubated with 5 mM fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone (24 h) (n = 4 at least). Relative protein levels of nuclear Nrf2 were normalized to LaminA, of total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M.. P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Article Snippet: Rabbit anti-Keap1 (#8047), mouse anti-Lamin A/C (#4777), rabbit anti-GST (#2625), mouse anti-NQO1 (#3187), rabbit anti-NLRP3 (#13158) and HRP-conjugated rabbit anti-IgG (#AP132P) were obtained from Cell Signaling Technology (Cambridge, USA).

    Techniques: Western Blot, Incubation, Transfection

    Polydatin attenuates fructose feeding-induced miR-200a low-expression, Keap1 up-regulation and Nrf2 antioxidant pathway inactivation in rats with liver oxidative stress. (A) qRT-PCR analysis of miR-200a expression levels in rat livers. Relative miR-200a levels were normalized to U6. (B) qRT-PCT analysis of Keap1 mRNA levels and Western blot analysis of Keap1 protein levels in rat livers. Relative mRNA levels of Keap1 were normalized to β-actin. Western blot analysis of total Nrf2 and nuclear Nrf2 (C), GST, HO-1 and NQO1 protein levels (D) in rat livers. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M. (n = 4 at least). P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Journal: Redox Biology

    Article Title: Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway

    doi: 10.1016/j.redox.2018.07.002

    Figure Lengend Snippet: Polydatin attenuates fructose feeding-induced miR-200a low-expression, Keap1 up-regulation and Nrf2 antioxidant pathway inactivation in rats with liver oxidative stress. (A) qRT-PCR analysis of miR-200a expression levels in rat livers. Relative miR-200a levels were normalized to U6. (B) qRT-PCT analysis of Keap1 mRNA levels and Western blot analysis of Keap1 protein levels in rat livers. Relative mRNA levels of Keap1 were normalized to β-actin. Western blot analysis of total Nrf2 and nuclear Nrf2 (C), GST, HO-1 and NQO1 protein levels (D) in rat livers. Relative protein levels of nuclear Nrf2 were normalized to LaminA, of Keap1, total Nrf2, GST, HO-1 and NQO1 were normalized to GAPDH or β-actin, respectively. All data are expressed as mean ± S.E.M. (n = 4 at least). P value was calculated by one-way ANOVA and further post hoc Dannelt testing. # P

    Article Snippet: Rabbit anti-Keap1 (#8047), mouse anti-Lamin A/C (#4777), rabbit anti-GST (#2625), mouse anti-NQO1 (#3187), rabbit anti-NLRP3 (#13158) and HRP-conjugated rabbit anti-IgG (#AP132P) were obtained from Cell Signaling Technology (Cambridge, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Induction of HO-1 is not protective against ATO-induced apoptosis in MM cell lines. U266, MM.1s, 8226/S and KMS11 were treated for 0, 6, 24, and 48 h with 2 μ m ATO. Total protein lysates were obtained and NQO1 ( A ) and HO-1 ( B ) protein expression

    Journal: The Journal of Biological Chemistry

    Article Title: Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-induced Antioxidant Response or Apoptosis *Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-induced Antioxidant Response or Apoptosis * S⃞

    doi: 10.1074/jbc.M806546200

    Figure Lengend Snippet: Induction of HO-1 is not protective against ATO-induced apoptosis in MM cell lines. U266, MM.1s, 8226/S and KMS11 were treated for 0, 6, 24, and 48 h with 2 μ m ATO. Total protein lysates were obtained and NQO1 ( A ) and HO-1 ( B ) protein expression

    Article Snippet: Antibodies —The following primary antibodies were used: rabbit anti-HO-1 polyclonal antibody (pAb) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-NQO1 monoclonal antibody (mAb) (Cell Signaling, Danvers, MA); rabbit anti-actin pAb (Sigma-Aldrich); rabbit anti-Nrf2 pAb (Santa Cruz Biotechnology); rabbit anti-Keap1 pAb (Proteintech Group Inc., Chicago, IL); rabbit anti-Lamin A/C pAb (Abcam, Cambridge, MA), and the mouse anti-Noxa mAb (Abcam).

    Techniques: Expressing

    (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, NQO1, and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p

    Journal: bioRxiv

    Article Title: An Evaluation of the In Vitro Roles and Mechanisms of Silibinin in Reducing Pyrazinamide- and Isoniazid-Induced Hepatocellular Damage

    doi: 10.1101/815241

    Figure Lengend Snippet: (color, 2-column). Silibinin induced expression of proteins in the Nrf2-ARE pathway and restored protein expression. Vehicle control was treated with 0.05% v/v DMSO. (A) In LO2, silibinin’s reduction of ROS levels when co-administered with I/P 40/10 was independent of HO-1 protein restoration. The administration of I/P 40/10 significantly reduced HO-1 levels without silibinin (one-way ANOVA, p = 0.0150), or with silibinin at 25 μM (one-way ANOVA, p = 0.0051) and 50 μM (one-way ANOVA, p = 0.0022). Silibinin alone did not induce the expression of Gclc, HO-1, NQO1, and Srxn1. Positive controls were treated with the Nrf2-ARE inducer CA 50 μM for 24 h. Data represent mean ± S.E.M. of three replicates. * p

    Article Snippet: Proteins were mixed with loading dye, boiled at 100°C for 5 min, separated on SDS-PAGE using 12% v/v polyacrylamide gels (Bio-Rad Laboratories, United States), then transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, United States) at 4°C with 100V for 2 h. Membranes were washed with Tris-buffered saline (1st Base, Singapore) containing 0.1% v/v Tween, blocked with 5% w/v bovine serum albumin (BSA), then incubated overnight at 4°C with the following primary antibodies in 2% w/v BSA: Rabbit anti-Gclc antibody (Abcam, United Kingdom; 1:1000); rabbit anti-NQO1 antibody (Cell Signalling, United States; 1:1000); rabbit anti-HO-1 antibody (Cell Signalling; United States; 1:1000); mouse anti-Srxn1 antibody (Santa Cruz, United States; 1:500); and mouse anti- β -actin antibody (Cell Signalling, United States; 1:10000).

    Techniques: Expressing

    (color, 2-column). INH, but not PZA, significantly suppressed HO-1 expression in LO2. INH suppressed HO-1 expression at 10 mM (one-way ANOVA, p = 0.0275), 20 mM (one-way ANOVA, p = 0.0133), and 40 mM (one-way ANOVA, p = 0.0028); but not Gclc, NQO1, and Srxn1 expression in LO2 in vitro . PZA did not have any effect on HO-1, Gclc, NQO1, and Srxn1 at the concentrations tested. Positive control was treated with 10 μM SU for 24 h. Data represent mean ± S.E.M. of two replicates. * p

    Journal: bioRxiv

    Article Title: An Evaluation of the In Vitro Roles and Mechanisms of Silibinin in Reducing Pyrazinamide- and Isoniazid-Induced Hepatocellular Damage

    doi: 10.1101/815241

    Figure Lengend Snippet: (color, 2-column). INH, but not PZA, significantly suppressed HO-1 expression in LO2. INH suppressed HO-1 expression at 10 mM (one-way ANOVA, p = 0.0275), 20 mM (one-way ANOVA, p = 0.0133), and 40 mM (one-way ANOVA, p = 0.0028); but not Gclc, NQO1, and Srxn1 expression in LO2 in vitro . PZA did not have any effect on HO-1, Gclc, NQO1, and Srxn1 at the concentrations tested. Positive control was treated with 10 μM SU for 24 h. Data represent mean ± S.E.M. of two replicates. * p

    Article Snippet: Proteins were mixed with loading dye, boiled at 100°C for 5 min, separated on SDS-PAGE using 12% v/v polyacrylamide gels (Bio-Rad Laboratories, United States), then transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, United States) at 4°C with 100V for 2 h. Membranes were washed with Tris-buffered saline (1st Base, Singapore) containing 0.1% v/v Tween, blocked with 5% w/v bovine serum albumin (BSA), then incubated overnight at 4°C with the following primary antibodies in 2% w/v BSA: Rabbit anti-Gclc antibody (Abcam, United Kingdom; 1:1000); rabbit anti-NQO1 antibody (Cell Signalling, United States; 1:1000); rabbit anti-HO-1 antibody (Cell Signalling; United States; 1:1000); mouse anti-Srxn1 antibody (Santa Cruz, United States; 1:500); and mouse anti- β -actin antibody (Cell Signalling, United States; 1:10000).

    Techniques: Expressing, In Vitro, Positive Control

    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of p38 MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 (NADPH quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of p38 MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 (NADPH quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Translocation Assay, Binding Assay

    A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histone H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histone H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Western Blot, Expressing

    A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histon H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histon H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Western Blot, Expressing