peg catalase  (Millipore)

 
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    Name:
    Catalase polyethylene glycol
    Description:

    Catalog Number:
    C4963
    Price:
    None
    Applications:
    Pulmonary artery endothelial cells treated with PEG-catalase showed effective inhibition of 20- HETE (hydroxyeicosatetraenoic acid)-induced increase in fluorescence. However, the experiment excludes potential nonspecific fluorescence of DCF (dichlorofluorescein). This experiment studied the effect of 20-HETE on superoxide production and NADPH oxidase activation.
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    Structured Review

    Millipore peg catalase
    Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of <t>PEG-SOD</t> (200 units mL −1 ) (A) or PEG-catalase (1000 units mL −1 ) (B) or <t>Sulindac</t> (40 mg·kg −1 day −1 for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10 −12 - 10 −8 mol·l −1 ). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.

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    peg catalase - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice"

    Article Title: Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice

    Journal: Kidney & blood pressure research

    doi: 10.1159/000490334

    Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of PEG-SOD (200 units mL −1 ) (A) or PEG-catalase (1000 units mL −1 ) (B) or Sulindac (40 mg·kg −1 day −1 for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10 −12 - 10 −8 mol·l −1 ). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.
    Figure Legend Snippet: Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of PEG-SOD (200 units mL −1 ) (A) or PEG-catalase (1000 units mL −1 ) (B) or Sulindac (40 mg·kg −1 day −1 for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10 −12 - 10 −8 mol·l −1 ). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.

    Techniques Used: Mouse Assay

    2) Product Images from "Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys"

    Article Title: Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00500.2020

    Acute polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) treatment improved autoregulatory behavior of afferent arterioles in ischemia-reperfused (IR) rats. A : the afferent arteriolar response to alterations in renal perfusion pressure was measured in male sham (circles, n = 7), IR (squares, n = 8), or IR + PEG-SOD (diamonds, n = 6) rats. PEG-SOD (100 U/mL) was delivered in the reconstituted perfusate blood, and the kidney was perfused for 20–25 min before the autoregulatory protocol was started. B : data were normalized as a percentage of the baseline diameter at 100 mmHg. Values are means ± SE. For within-group analysis, one-way repeated-measures ANOVA with a Dunnett’s post hoc test was performed. * P
    Figure Legend Snippet: Acute polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) treatment improved autoregulatory behavior of afferent arterioles in ischemia-reperfused (IR) rats. A : the afferent arteriolar response to alterations in renal perfusion pressure was measured in male sham (circles, n = 7), IR (squares, n = 8), or IR + PEG-SOD (diamonds, n = 6) rats. PEG-SOD (100 U/mL) was delivered in the reconstituted perfusate blood, and the kidney was perfused for 20–25 min before the autoregulatory protocol was started. B : data were normalized as a percentage of the baseline diameter at 100 mmHg. Values are means ± SE. For within-group analysis, one-way repeated-measures ANOVA with a Dunnett’s post hoc test was performed. * P

    Techniques Used:

    Acute polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) or polyethylene glycol-conjugated catalase (PEG-catalase) treatment did not alter afferent arteriolar autoregulatory profile in sham rats. A : the afferent arteriolar response to alterations in renal perfusion pressure was measured in male sham (circles, n = 7), sham + PEG-SOD (squares, n = 6), or sham + PEG-catalase (diamonds, n = 5) rats. PEG-SOD (100 U/mL) or PEG-catalase (1,000 U/mL) was delivered in the reconstituted perfusate blood, and the kidney was perfused for 20–25 min before the autoregulatory protocol was started. B : data were normalized as a percentage of the baseline diameter at 100 mmHg. Values are means ± SE. For within-group analysis, one-way repeated-measures ANOVA with a Dunnett’s post hoc test was performed. * P
    Figure Legend Snippet: Acute polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) or polyethylene glycol-conjugated catalase (PEG-catalase) treatment did not alter afferent arteriolar autoregulatory profile in sham rats. A : the afferent arteriolar response to alterations in renal perfusion pressure was measured in male sham (circles, n = 7), sham + PEG-SOD (squares, n = 6), or sham + PEG-catalase (diamonds, n = 5) rats. PEG-SOD (100 U/mL) or PEG-catalase (1,000 U/mL) was delivered in the reconstituted perfusate blood, and the kidney was perfused for 20–25 min before the autoregulatory protocol was started. B : data were normalized as a percentage of the baseline diameter at 100 mmHg. Values are means ± SE. For within-group analysis, one-way repeated-measures ANOVA with a Dunnett’s post hoc test was performed. * P

    Techniques Used:

    3) Product Images from "α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction"

    Article Title: α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvy172

    Vascular oxidative stress is increased in mice lacking myelomonocytic cell α1AMPK. ( A ) Aortic 3-nitrotyrosine staining was used as a surrogate for peroxynitrite formation and assessed by dot blot technique. Pooled samples ( n = 3) of n = 6 mice. ( B ) Vascular superoxide anion production was analysed by dihydroethidium staining of aortic cryosections. ( C ) Measurement of hydrogen peroxide formation in aortic tissue was performed using an amplex red-based HPLC assay ( n = 4–7). Statistical significance was calculated by one-way ANOVA and Tukey multiple comparison test ( A and C ). ( D ) Aortic rings from myelomonocytic cell-specific α1AMPK knockout mice (α1AMPK fl/fl x LysM-Cre + ) and corresponding wild-type littermates (LysM Cre/wt ) treated with ATII (1.0 mg/kg for 7 days) were incubated with PEG-SOD ex vivo ; endothelial-dependent relaxation in response to acetylcholine (ACh) was analysed by isometric tension studies ( n = 8 animals/group). ( E ) Phorbol ester (PDBu) stimulated oxidative burst in whole blood was determined by L-012 enhanced chemiluminescence ( n = 12). Statistical significance was calculated by two-way ANOVA and Tukey post hoc test (for detailed P , Figures S8 and S9 ). ( F ) Nox2 mRNA ( n = 4–6) and protein expression in aortic tissue. Pooled samples ( n = 3) of n = 6 mice. One-way ANOVA and Tukey multiple comparison test were used to calculate statistical significance. * P
    Figure Legend Snippet: Vascular oxidative stress is increased in mice lacking myelomonocytic cell α1AMPK. ( A ) Aortic 3-nitrotyrosine staining was used as a surrogate for peroxynitrite formation and assessed by dot blot technique. Pooled samples ( n = 3) of n = 6 mice. ( B ) Vascular superoxide anion production was analysed by dihydroethidium staining of aortic cryosections. ( C ) Measurement of hydrogen peroxide formation in aortic tissue was performed using an amplex red-based HPLC assay ( n = 4–7). Statistical significance was calculated by one-way ANOVA and Tukey multiple comparison test ( A and C ). ( D ) Aortic rings from myelomonocytic cell-specific α1AMPK knockout mice (α1AMPK fl/fl x LysM-Cre + ) and corresponding wild-type littermates (LysM Cre/wt ) treated with ATII (1.0 mg/kg for 7 days) were incubated with PEG-SOD ex vivo ; endothelial-dependent relaxation in response to acetylcholine (ACh) was analysed by isometric tension studies ( n = 8 animals/group). ( E ) Phorbol ester (PDBu) stimulated oxidative burst in whole blood was determined by L-012 enhanced chemiluminescence ( n = 12). Statistical significance was calculated by two-way ANOVA and Tukey post hoc test (for detailed P , Figures S8 and S9 ). ( F ) Nox2 mRNA ( n = 4–6) and protein expression in aortic tissue. Pooled samples ( n = 3) of n = 6 mice. One-way ANOVA and Tukey multiple comparison test were used to calculate statistical significance. * P

    Techniques Used: Mouse Assay, Staining, Dot Blot, High Performance Liquid Chromatography, Knock-Out, Incubation, Ex Vivo, Expressing

    4) Product Images from "BCR-ABL tyrosine kinase inhibitors promote pathological changes in dilator phenotype in the human microvasculature"

    Article Title: BCR-ABL tyrosine kinase inhibitors promote pathological changes in dilator phenotype in the human microvasculature

    Journal: Microcirculation (New York, N.Y. : 1994)

    doi: 10.1111/micc.12625

    Flow-mediated dilation in the presence or absence of either L-NAME or PEG-Catalase. Panel A. Consistent with our previous work, in arterioles incubated in PBS vehicle, the nitric oxide synthase inhibitor L-NAME (n = 5) reduced FMD compared to control (n = 8) while the hydrogen peroxide scavenger PEG-Catalase had no effect on FMD (n = 7). In arterioles incubated with either imatinib (Panel B) or nilotinib (Panel C), the nitric oxide synthase inhibitor L-NAME (n = 7.4) had no effect on FMD compared to control (n = 7.7) while the hydrogen peroxide scavenger PEG-Catalase reduced FMD (n = 7.5). Area under the curve analysis is shown in Panel D. In imatinib- and nilotinib-treated vessels, the area under the curve for PEG-Catalase–inhibitable dilation was reduced compared to control. All data shown as mean ± SD. * P
    Figure Legend Snippet: Flow-mediated dilation in the presence or absence of either L-NAME or PEG-Catalase. Panel A. Consistent with our previous work, in arterioles incubated in PBS vehicle, the nitric oxide synthase inhibitor L-NAME (n = 5) reduced FMD compared to control (n = 8) while the hydrogen peroxide scavenger PEG-Catalase had no effect on FMD (n = 7). In arterioles incubated with either imatinib (Panel B) or nilotinib (Panel C), the nitric oxide synthase inhibitor L-NAME (n = 7.4) had no effect on FMD compared to control (n = 7.7) while the hydrogen peroxide scavenger PEG-Catalase reduced FMD (n = 7.5). Area under the curve analysis is shown in Panel D. In imatinib- and nilotinib-treated vessels, the area under the curve for PEG-Catalase–inhibitable dilation was reduced compared to control. All data shown as mean ± SD. * P

    Techniques Used: Incubation

    5) Product Images from "α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction"

    Article Title: α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvy172

    Vascular oxidative stress is increased in mice lacking myelomonocytic cell α1AMPK. ( A ) Aortic 3-nitrotyrosine staining was used as a surrogate for peroxynitrite formation and assessed by dot blot technique. Pooled samples ( n = 3) of n = 6 mice. ( B ) Vascular superoxide anion production was analysed by dihydroethidium staining of aortic cryosections. ( C ) Measurement of hydrogen peroxide formation in aortic tissue was performed using an amplex red-based HPLC assay ( n = 4–7). Statistical significance was calculated by one-way ANOVA and Tukey multiple comparison test ( A and C ). ( D ) Aortic rings from myelomonocytic cell-specific α1AMPK knockout mice (α1AMPK fl/fl x LysM-Cre + ) and corresponding wild-type littermates (LysM Cre/wt ) treated with ATII (1.0 mg/kg for 7 days) were incubated with PEG-SOD ex vivo ; endothelial-dependent relaxation in response to acetylcholine (ACh) was analysed by isometric tension studies ( n = 8 animals/group). ( E ) Phorbol ester (PDBu) stimulated oxidative burst in whole blood was determined by L-012 enhanced chemiluminescence ( n = 12). Statistical significance was calculated by two-way ANOVA and Tukey post hoc test (for detailed P , Figures S8 and S9 ). ( F ) Nox2 mRNA ( n = 4–6) and protein expression in aortic tissue. Pooled samples ( n = 3) of n = 6 mice. One-way ANOVA and Tukey multiple comparison test were used to calculate statistical significance. * P
    Figure Legend Snippet: Vascular oxidative stress is increased in mice lacking myelomonocytic cell α1AMPK. ( A ) Aortic 3-nitrotyrosine staining was used as a surrogate for peroxynitrite formation and assessed by dot blot technique. Pooled samples ( n = 3) of n = 6 mice. ( B ) Vascular superoxide anion production was analysed by dihydroethidium staining of aortic cryosections. ( C ) Measurement of hydrogen peroxide formation in aortic tissue was performed using an amplex red-based HPLC assay ( n = 4–7). Statistical significance was calculated by one-way ANOVA and Tukey multiple comparison test ( A and C ). ( D ) Aortic rings from myelomonocytic cell-specific α1AMPK knockout mice (α1AMPK fl/fl x LysM-Cre + ) and corresponding wild-type littermates (LysM Cre/wt ) treated with ATII (1.0 mg/kg for 7 days) were incubated with PEG-SOD ex vivo ; endothelial-dependent relaxation in response to acetylcholine (ACh) was analysed by isometric tension studies ( n = 8 animals/group). ( E ) Phorbol ester (PDBu) stimulated oxidative burst in whole blood was determined by L-012 enhanced chemiluminescence ( n = 12). Statistical significance was calculated by two-way ANOVA and Tukey post hoc test (for detailed P , Figures S8 and S9 ). ( F ) Nox2 mRNA ( n = 4–6) and protein expression in aortic tissue. Pooled samples ( n = 3) of n = 6 mice. One-way ANOVA and Tukey multiple comparison test were used to calculate statistical significance. * P

    Techniques Used: Mouse Assay, Staining, Dot Blot, High Performance Liquid Chromatography, Knock-Out, Incubation, Ex Vivo, Expressing

    6) Product Images from "Role of Mitochondrial Electron Transport Chain Complexes in Capsaicin Mediated Oxidative Stress Leading to Apoptosis in Pancreatic Cancer Cells"

    Article Title: Role of Mitochondrial Electron Transport Chain Complexes in Capsaicin Mediated Oxidative Stress Leading to Apoptosis in Pancreatic Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020151

    Anti-oxidants prevent capsaicin-induced apoptosis. ( A ) BxPC-3 cells were pretreated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h and then treated with DMSO or 150 µM capsaicin for 24 h, ( B ) BxPC-3 ρ 0 cells were treated with DMSO or 150 µM capsaicin for 24 h, and apoptosis was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 3) of three independent experiments. *Statistically different when compared with control ( P
    Figure Legend Snippet: Anti-oxidants prevent capsaicin-induced apoptosis. ( A ) BxPC-3 cells were pretreated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h and then treated with DMSO or 150 µM capsaicin for 24 h, ( B ) BxPC-3 ρ 0 cells were treated with DMSO or 150 µM capsaicin for 24 h, and apoptosis was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 3) of three independent experiments. *Statistically different when compared with control ( P

    Techniques Used: Flow Cytometry

    7) Product Images from "The NADPH Oxidase Subunit p22phox Inhibits the Function of the Tumor Suppressor Protein Tuberin"

    Article Title: The NADPH Oxidase Subunit p22phox Inhibits the Function of the Tumor Suppressor Protein Tuberin

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090606

    Superoxide is the major ROS involved in maintaining HIF-2α in the absence of VHL. A: Where indicated, antioxidants targeting hydrogen peroxide, PEG-Catalase (500 U/ml), NAC (100 mmol/L), or vehicle alone (−) were added to RCC 786-O cells
    Figure Legend Snippet: Superoxide is the major ROS involved in maintaining HIF-2α in the absence of VHL. A: Where indicated, antioxidants targeting hydrogen peroxide, PEG-Catalase (500 U/ml), NAC (100 mmol/L), or vehicle alone (−) were added to RCC 786-O cells

    Techniques Used:

    8) Product Images from "TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase"

    Article Title: TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200318696

    Augmented LPS induction of TLR2 in endothelial cells requires PMN NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91 phox–/– mice, respectively, and were added at a concentration of 1 × 10 5 cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis ( a ) as well as Western blotting with anti-TLR2 antibody ( b ). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis ( a and b , lane 6). ( c ) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47 phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable PEG-catalase (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.
    Figure Legend Snippet: Augmented LPS induction of TLR2 in endothelial cells requires PMN NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91 phox–/– mice, respectively, and were added at a concentration of 1 × 10 5 cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis ( a ) as well as Western blotting with anti-TLR2 antibody ( b ). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis ( a and b , lane 6). ( c ) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47 phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable PEG-catalase (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.

    Techniques Used: Expressing, Isolation, Cell Culture, Mouse Assay, Concentration Assay, Incubation, Lysis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay

    9) Product Images from "2-Deoxy-d-Glucose Combined with Cisplatin Enhances Cytotoxicity via Metabolic Oxidative Stress in Human Head and Neck Cancer Cells"

    Article Title: 2-Deoxy-d-Glucose Combined with Cisplatin Enhances Cytotoxicity via Metabolic Oxidative Stress in Human Head and Neck Cancer Cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-06-3717

    Effect of PEG-SOD and PEG-catalase on 2DG toxicity in FaDu cells. Cells were treated with 18 μmol/L PEG, 100 units/mL PEG-SOD, 100 units/mL PEG-catalase ( PEG-CAT ), or 50 units/mL PEG-SOD + 50 units/mL PEG-catalase for 1 h before and during treatment
    Figure Legend Snippet: Effect of PEG-SOD and PEG-catalase on 2DG toxicity in FaDu cells. Cells were treated with 18 μmol/L PEG, 100 units/mL PEG-SOD, 100 units/mL PEG-catalase ( PEG-CAT ), or 50 units/mL PEG-SOD + 50 units/mL PEG-catalase for 1 h before and during treatment

    Techniques Used:

    10) Product Images from "Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds"

    Article Title: Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201209077

    VL are enriched in and functionally dependent on NADPH oxidase activity. (a) MVECs coexpressing p47phox-DsRed and actin-GFP were imaged live during T cell diapedesis. p47phox was enriched in ventral (IRM) actin-rich initiation nodes (N) and in VL leading edge (arrowheads). See Video 10 . (b) Representative ratiometric images of H 2 O 2 production in (i) pore- and (ii) gap-closing VL in MVECs expressing the biosensor mHyPer during T cell diapedesis. (c) Quantification of p47phox (i) and H 2 O 2 (ii) enrichment in VL. n > 13. (d, i) Representative NADPH oxidase inhibition/washout imaging experiment. After multiple T cell diapedesis events on MVECs expressing mYFP, apocynin was added. Arrows indicate multiple pores and gaps that persisted for 15 min, but were rapidly healed by steered VL (arrowheads) after drug washout. See Video 10. Experiments as in d (i) were performed with VAS-2870, DPI, PEG-catalase, and DMSO (control) and quantified by cumulative wound closure analysis, shown as a continuous line plot (ii) and bar graph of single pre/post-washout time points (iii). n > 5. (e) MVECs coexpressing mDsRed and mHyPer were imaged during probe wounds and after addition of VAS-2870. Representative mDsRed images (i), kymograph (ii), mHyper ratiometic images (iii), and quantitation (iv) are shown. n = 3. Values represent mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P
    Figure Legend Snippet: VL are enriched in and functionally dependent on NADPH oxidase activity. (a) MVECs coexpressing p47phox-DsRed and actin-GFP were imaged live during T cell diapedesis. p47phox was enriched in ventral (IRM) actin-rich initiation nodes (N) and in VL leading edge (arrowheads). See Video 10 . (b) Representative ratiometric images of H 2 O 2 production in (i) pore- and (ii) gap-closing VL in MVECs expressing the biosensor mHyPer during T cell diapedesis. (c) Quantification of p47phox (i) and H 2 O 2 (ii) enrichment in VL. n > 13. (d, i) Representative NADPH oxidase inhibition/washout imaging experiment. After multiple T cell diapedesis events on MVECs expressing mYFP, apocynin was added. Arrows indicate multiple pores and gaps that persisted for 15 min, but were rapidly healed by steered VL (arrowheads) after drug washout. See Video 10. Experiments as in d (i) were performed with VAS-2870, DPI, PEG-catalase, and DMSO (control) and quantified by cumulative wound closure analysis, shown as a continuous line plot (ii) and bar graph of single pre/post-washout time points (iii). n > 5. (e) MVECs coexpressing mDsRed and mHyPer were imaged during probe wounds and after addition of VAS-2870. Representative mDsRed images (i), kymograph (ii), mHyper ratiometic images (iii), and quantitation (iv) are shown. n = 3. Values represent mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P

    Techniques Used: Activity Assay, Expressing, Inhibition, Imaging, Quantitation Assay

    11) Product Images from "Increased mitochondrial ROS generation mediates the loss of the anti‐contractile effects of perivascular adipose tissue in high‐fat diet obese mice) Increased mitochondrial ROS generation mediates the loss of the anti‐contractile effects of perivascular adipose tissue in high‐fat diet obese mice"

    Article Title: Increased mitochondrial ROS generation mediates the loss of the anti‐contractile effects of perivascular adipose tissue in high‐fat diet obese mice) Increased mitochondrial ROS generation mediates the loss of the anti‐contractile effects of perivascular adipose tissue in high‐fat diet obese mice

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13687

    Mitochondria are a potential source of increased ROS in PVAT from obese mice, and mitochondria‐derived ROS alter the expression of antioxidant enzymes in PVAT. Concentration–effect curves to PE were performed in endothelium‐denuded aortic rings from control [( A and C ) n = 8 for each experimental group] and obese [( B and D ) n = 8 for each experimental group] mice. The role of mROS on PVAT modulation of aortic smooth muscle contraction was investigated using MnTMPyP (3 × 10 −5 M), a mitochondria‐targeted superoxide scavenger and PEG‐catalase (Peg‐cat; 200 U·mL −1 ), which dismutates mitochondria‐derived H 2 O 2 . Protein expression and activity of the antioxidant enzymes SOD‐Mn [( E and G ) n = 5 in both groups] and catalase [( F and H ) n = 5 in both groups] were determined by Western blot and SOD‐Mn and catalase activity assay kits, respectively, in PVAT from control and obese mice. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β‐actin expression and are expressed as relative units. Results represent the mean ± SEM. * P
    Figure Legend Snippet: Mitochondria are a potential source of increased ROS in PVAT from obese mice, and mitochondria‐derived ROS alter the expression of antioxidant enzymes in PVAT. Concentration–effect curves to PE were performed in endothelium‐denuded aortic rings from control [( A and C ) n = 8 for each experimental group] and obese [( B and D ) n = 8 for each experimental group] mice. The role of mROS on PVAT modulation of aortic smooth muscle contraction was investigated using MnTMPyP (3 × 10 −5 M), a mitochondria‐targeted superoxide scavenger and PEG‐catalase (Peg‐cat; 200 U·mL −1 ), which dismutates mitochondria‐derived H 2 O 2 . Protein expression and activity of the antioxidant enzymes SOD‐Mn [( E and G ) n = 5 in both groups] and catalase [( F and H ) n = 5 in both groups] were determined by Western blot and SOD‐Mn and catalase activity assay kits, respectively, in PVAT from control and obese mice. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β‐actin expression and are expressed as relative units. Results represent the mean ± SEM. * P

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

    12) Product Images from "Chronic hypoxia limits H2O2-induced inhibition of ASIC1-dependent store-operated calcium entry in pulmonary arterial smooth muscle"

    Article Title: Chronic hypoxia limits H2O2-induced inhibition of ASIC1-dependent store-operated calcium entry in pulmonary arterial smooth muscle

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00095.2014

    CH increases pulmonary arterial antioxidant capacity. A : Amplex Red RFU from control intrapulmonary arteries following treatment with PEG-SOD (50 U/ml), the SOD mimetic, tiron (10 mM), or inhibition of catalase and glutathione peroxidase (GPx) with 3-amino-1,2,4-triazole (AT; 5 mM) and mercaptosuccinic acid (MSA; 3 mM) are within the linear range of the background-subtracted standard curve. B : summary data of H 2 O 2 levels in intrapulmonary arteries from control and CH rats following above treatments. C : rate of H 2 O 2 catalysis (1 μM) by control and CH intrapulmonary arteries measured by Amplex Red fluorescence. Values are means ± SE; n = 6–8 animals per group; τ P
    Figure Legend Snippet: CH increases pulmonary arterial antioxidant capacity. A : Amplex Red RFU from control intrapulmonary arteries following treatment with PEG-SOD (50 U/ml), the SOD mimetic, tiron (10 mM), or inhibition of catalase and glutathione peroxidase (GPx) with 3-amino-1,2,4-triazole (AT; 5 mM) and mercaptosuccinic acid (MSA; 3 mM) are within the linear range of the background-subtracted standard curve. B : summary data of H 2 O 2 levels in intrapulmonary arteries from control and CH rats following above treatments. C : rate of H 2 O 2 catalysis (1 μM) by control and CH intrapulmonary arteries measured by Amplex Red fluorescence. Values are means ± SE; n = 6–8 animals per group; τ P

    Techniques Used: Inhibition, Fluorescence

    Di-(4-carboxybenzyl) hyponitrite (SOTS-1) increases PASMC O 2 ·− levels. Representative images ( A ) and summary data ( B ) showing background-subtracted MFI of DHE in PASMC from control rats treated with increasing concentrations of SOTS-1 (0.01, 0.1, 1.0 mM) or SOTS-1 (0.01 mM) plus PEG-SOD (50 U/ml) or PEG-catalase (250 U/ml). Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. Values are means ± SE; n = 3–5 animals per group. ** P
    Figure Legend Snippet: Di-(4-carboxybenzyl) hyponitrite (SOTS-1) increases PASMC O 2 ·− levels. Representative images ( A ) and summary data ( B ) showing background-subtracted MFI of DHE in PASMC from control rats treated with increasing concentrations of SOTS-1 (0.01, 0.1, 1.0 mM) or SOTS-1 (0.01 mM) plus PEG-SOD (50 U/ml) or PEG-catalase (250 U/ml). Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. Values are means ± SE; n = 3–5 animals per group. ** P

    Techniques Used: Fluorescence, Immunofluorescence

    13) Product Images from "Role of ER Stress Response in Photodynamic Therapy: ROS Generated in Different Subcellular Compartments Trigger Diverse Cell Death Pathways"

    Article Title: Role of ER Stress Response in Photodynamic Therapy: ROS Generated in Different Subcellular Compartments Trigger Diverse Cell Death Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032972

    Role of ROS generation in EG-porphyrin-mediated cell death. ( A ) Effect ROS scavengers on cell viability after PDT. 4T1 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then pre-incubated for 1 h with L-histidine (20 mM), trolox (5 mM), NAC (15 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM) before light exposure. Similarly, HL60 cells were pre-incubated with L-histidine (20 mM), trolox (4 mM), NAC (5 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM). After irradiation the cells were further cultivated and cell viability was determined 24 h post-PDT by the trypan blue exclusion method. Cells treated with EG-porphyrin derivatives without irradiation were used as controls (dark). The percentage of dead cells was expressed as the mean ± SD (n = 3). *P
    Figure Legend Snippet: Role of ROS generation in EG-porphyrin-mediated cell death. ( A ) Effect ROS scavengers on cell viability after PDT. 4T1 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then pre-incubated for 1 h with L-histidine (20 mM), trolox (5 mM), NAC (15 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM) before light exposure. Similarly, HL60 cells were pre-incubated with L-histidine (20 mM), trolox (4 mM), NAC (5 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM). After irradiation the cells were further cultivated and cell viability was determined 24 h post-PDT by the trypan blue exclusion method. Cells treated with EG-porphyrin derivatives without irradiation were used as controls (dark). The percentage of dead cells was expressed as the mean ± SD (n = 3). *P

    Techniques Used: Incubation, Irradiation

    14) Product Images from "Pressor Effect of Apelin-13 in the Rostral Ventrolateral Medulla: Role of NAD(P)H Oxidase-Derived Superoxide"

    Article Title: Pressor Effect of Apelin-13 in the Rostral Ventrolateral Medulla: Role of NAD(P)H Oxidase-Derived Superoxide

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.110.174102

    Identification of ROS involved in the chronotropic actions of apelin-13 in neurons. A, role of superoxide. The neuronal firing rate was recorded in neurons treated under the following conditions: superfusion of PBS control (Con); superfusion of apelin-13 (APLN, 100 nM) followed by washout of apelin-13 with fresh superfusate solution; superfusion of PEG-SOD (SOD, 25 U/ml), and PEG-SOD plus apelin-13 (SOD+APLN). Data are mean ± S.E. ( n = 9 neurons). B, effect of xanthine-xanthine oxidase on neuronal firing rate. The neuronal firing rate was recorded in neurons treated under PBS control (Con) and superfusion of xanthine-xanthine oxidase (10 mM and 20 mU/ml). Data are mean ± S.E. ( n = 5 neurons). C, role of hydrogen peroxide. The neuronal firing rate was recorded in neurons treated under the following conditions: superfusion of PBS control (Con); superfusion of apelin-13 (APLN, 100 nM) followed by washout of apelin-13 with fresh superfusate solution, superfusion of PEG-catalase (CAT, 250 U/ml), and PEG-catalase plus apelin-13 (CAT+APLN). Data are mean ± S.E. ( n = 7 neurons). D, effect of hydrogen peroxide on neuronal firing rate. Data are mean ± S.E. of neuronal firing rate in eight neurons treated under PBS control (Con) and superfusion of hydrogen peroxide (H 2 O 2 , 1 μM). *, P
    Figure Legend Snippet: Identification of ROS involved in the chronotropic actions of apelin-13 in neurons. A, role of superoxide. The neuronal firing rate was recorded in neurons treated under the following conditions: superfusion of PBS control (Con); superfusion of apelin-13 (APLN, 100 nM) followed by washout of apelin-13 with fresh superfusate solution; superfusion of PEG-SOD (SOD, 25 U/ml), and PEG-SOD plus apelin-13 (SOD+APLN). Data are mean ± S.E. ( n = 9 neurons). B, effect of xanthine-xanthine oxidase on neuronal firing rate. The neuronal firing rate was recorded in neurons treated under PBS control (Con) and superfusion of xanthine-xanthine oxidase (10 mM and 20 mU/ml). Data are mean ± S.E. ( n = 5 neurons). C, role of hydrogen peroxide. The neuronal firing rate was recorded in neurons treated under the following conditions: superfusion of PBS control (Con); superfusion of apelin-13 (APLN, 100 nM) followed by washout of apelin-13 with fresh superfusate solution, superfusion of PEG-catalase (CAT, 250 U/ml), and PEG-catalase plus apelin-13 (CAT+APLN). Data are mean ± S.E. ( n = 7 neurons). D, effect of hydrogen peroxide on neuronal firing rate. Data are mean ± S.E. of neuronal firing rate in eight neurons treated under PBS control (Con) and superfusion of hydrogen peroxide (H 2 O 2 , 1 μM). *, P

    Techniques Used:

    15) Product Images from "Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production"

    Article Title: Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production

    Journal: Cardiovascular Diabetology

    doi: 10.1186/1475-2840-11-5

    Effect of high-fat diet in the generation of intraplatelet reactive-oxygen species (ROS) . Male Wistar rats were fed with either a standard chow diet (SCD) or high-fat diet (HFD) during 10 weeks. Washed platelets (1.2 × 10 8 platelets/ml) from SCD or HFF rats were pre-incubated with N-acetylcysteine (NAC, 1 mM for 15 min) or PEG-catalase (1000 mU/ml, 15 min) and then stimulated with ADP (10 μM). Generation of ROS was quantified by flow cytometry using 2'-7'-dichloroflurescin diacetate (DCFH-DA). Results are shown as mean ± SEM values for n = 6-7. * P
    Figure Legend Snippet: Effect of high-fat diet in the generation of intraplatelet reactive-oxygen species (ROS) . Male Wistar rats were fed with either a standard chow diet (SCD) or high-fat diet (HFD) during 10 weeks. Washed platelets (1.2 × 10 8 platelets/ml) from SCD or HFF rats were pre-incubated with N-acetylcysteine (NAC, 1 mM for 15 min) or PEG-catalase (1000 mU/ml, 15 min) and then stimulated with ADP (10 μM). Generation of ROS was quantified by flow cytometry using 2'-7'-dichloroflurescin diacetate (DCFH-DA). Results are shown as mean ± SEM values for n = 6-7. * P

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    Effect of N-Acetylcysteine (NAC) and PEG-catalase on washed platelet aggregation of rats fed with a standard chow diet (SCD) or high-fat diet (HFD) during 10 weeks . Washed platelets (1.2 × 10 8 platelets/ml) from SCD or HFD rats were stimulated with thrombin (100 mU/ml; panel A) or ADP (10 μM; panel B) in the absence or the presence of NAC (1 mM) or PEG-catalase (1000 U/ml). Results are shown as mean ± SEM values ( n = 4-7). * P
    Figure Legend Snippet: Effect of N-Acetylcysteine (NAC) and PEG-catalase on washed platelet aggregation of rats fed with a standard chow diet (SCD) or high-fat diet (HFD) during 10 weeks . Washed platelets (1.2 × 10 8 platelets/ml) from SCD or HFD rats were stimulated with thrombin (100 mU/ml; panel A) or ADP (10 μM; panel B) in the absence or the presence of NAC (1 mM) or PEG-catalase (1000 U/ml). Results are shown as mean ± SEM values ( n = 4-7). * P

    Techniques Used:

    16) Product Images from "Reactive Oxygen Species Are Involved in Regulating Hypocontractility of Mesenteric Artery to Norepinephrine in Cirrhotic Rats with Portal Hypertension"

    Article Title: Reactive Oxygen Species Are Involved in Regulating Hypocontractility of Mesenteric Artery to Norepinephrine in Cirrhotic Rats with Portal Hypertension

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.8081

    Dose-response curves of isolated mesenteric artery contractility to norepinephrine with different concentrations. Vasoconstriction rate and the logarithm of norepinephrine concentration were used as vertical axis and abscissa, respectively. The results showed that dose-response curve of mesenteric arteriole contractility to norepinephrine moved to the left and EC 50 decreased in rats with cirrhosis in apocynin ( A, B ) or PEG-catalase ( C, D ) treatment. A: * PHTA compared with PHTC, P
    Figure Legend Snippet: Dose-response curves of isolated mesenteric artery contractility to norepinephrine with different concentrations. Vasoconstriction rate and the logarithm of norepinephrine concentration were used as vertical axis and abscissa, respectively. The results showed that dose-response curve of mesenteric arteriole contractility to norepinephrine moved to the left and EC 50 decreased in rats with cirrhosis in apocynin ( A, B ) or PEG-catalase ( C, D ) treatment. A: * PHTA compared with PHTC, P

    Techniques Used: Isolation, Concentration Assay

    Fluorescence intensity of vascular wall in rats after drugs treatment. ( A ) Fluorescence intensity was the strongest in cirrhotic rats and diminished by treatment with apocynin, tempol or PEG-catalase. Each image is representative of results from 6 different animals. ( B ) The quantitative of fluorescence intensity was analyzed in rats after treatment with different drugs. Values are mean ± SEM; n=6 for each group. **: compared with NC, P
    Figure Legend Snippet: Fluorescence intensity of vascular wall in rats after drugs treatment. ( A ) Fluorescence intensity was the strongest in cirrhotic rats and diminished by treatment with apocynin, tempol or PEG-catalase. Each image is representative of results from 6 different animals. ( B ) The quantitative of fluorescence intensity was analyzed in rats after treatment with different drugs. Values are mean ± SEM; n=6 for each group. **: compared with NC, P

    Techniques Used: Fluorescence

    17) Product Images from "Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice"

    Article Title: Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice

    Journal: Kidney & blood pressure research

    doi: 10.1159/000490334

    Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of PEG-SOD (200 units mL −1 ) (A) or PEG-catalase (1000 units mL −1 ) (B) or Sulindac (40 mg·kg −1 day −1 for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10 −12 - 10 −8 mol·l −1 ). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.
    Figure Legend Snippet: Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of PEG-SOD (200 units mL −1 ) (A) or PEG-catalase (1000 units mL −1 ) (B) or Sulindac (40 mg·kg −1 day −1 for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10 −12 - 10 −8 mol·l −1 ). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.

    Techniques Used: Mouse Assay

    18) Product Images from "Hydrogen Peroxide Regulates Extracellular Superoxide Dismutase Activity and Expression in Neonatal Pulmonary Hypertension"

    Article Title: Hydrogen Peroxide Regulates Extracellular Superoxide Dismutase Activity and Expression in Neonatal Pulmonary Hypertension

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2010.3630

    PEG-catalase decreases superoxide levels in resistance PAs. (A) Frozen lung sections from PPHN lambs ventilated for 24 h with 100% oxygen alone (L100; n = 7) or in combination with 20,000 U/kg PEG-catalase administered
    Figure Legend Snippet: PEG-catalase decreases superoxide levels in resistance PAs. (A) Frozen lung sections from PPHN lambs ventilated for 24 h with 100% oxygen alone (L100; n = 7) or in combination with 20,000 U/kg PEG-catalase administered

    Techniques Used:

    PEG-catalase decreases ecSOD expression but increases activity in PPHN lungs. Lung tissue was harvested from fetal control and fetal PPHN lambs ( n = 6 each), and from PPHN lambs ventilated for 24 h with 100% O 2 alone (L100; n = 7)
    Figure Legend Snippet: PEG-catalase decreases ecSOD expression but increases activity in PPHN lungs. Lung tissue was harvested from fetal control and fetal PPHN lambs ( n = 6 each), and from PPHN lambs ventilated for 24 h with 100% O 2 alone (L100; n = 7)

    Techniques Used: Expressing, Activity Assay

    PEG-catalase restores ecSOD activity in PPHN PASMCs. PASMCs isolated from PPHN lambs were treated with 0–200 U/ml PEG-catalase for 24 h in serum-free medium. ecSOD protein was purified from cell extracts with affinity chromatography,
    Figure Legend Snippet: PEG-catalase restores ecSOD activity in PPHN PASMCs. PASMCs isolated from PPHN lambs were treated with 0–200 U/ml PEG-catalase for 24 h in serum-free medium. ecSOD protein was purified from cell extracts with affinity chromatography,

    Techniques Used: Activity Assay, Isolation, Purification, Affinity Chromatography

    PEG-catalase improves the arterial-to-alveolar oxygenation (a/A) ratio in PPHN lambs. The a/A ratio was calculated over a 24-h period for lambs with PPHN ventilated with 100% oxygen alone ( n = 12) or with 100% oxygen in combination with
    Figure Legend Snippet: PEG-catalase improves the arterial-to-alveolar oxygenation (a/A) ratio in PPHN lambs. The a/A ratio was calculated over a 24-h period for lambs with PPHN ventilated with 100% oxygen alone ( n = 12) or with 100% oxygen in combination with

    Techniques Used:

    19) Product Images from "Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells"

    Article Title: Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2016/4941623

    JC induces apoptosis by increasing intracellular ROS. (a, c) Effect of JC on the total ROS levels in HepG2 and BEL-7402 cells was examined by flow cytometry. HepG2 cells were treated with DMSO or 8 μ g/mL JC for 12, 24, 36, and 48 hours (a). BEL-7402 cells were treated with DMSO or 6.7, 8.7, and 10.5 μ g/mL of JC for 12 hours (c). (b) Effect of JC on superoxide anion levels in HepG2 cells was examined by flow cytometry. The levels of ROS in HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for 12, 24, 36, and 48 hours and analyzed by flow cytometry. (d–f) Effects of JC on catalase and SOD2 expression and Akt activation were determined by Western blot analysis. HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for the indicated times (d and e). BEL-7402 cells were treated with DMSO or 8.7 μ g/mL of JC for the indicated times (f). GAPDH was used as a loading control. (g–j) NAC and PEG-catalase abrogate JC-induced apoptosis. HepG2 cells were pretreated with NAC and PEG-catalase for 1 hour. HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for 36 hours and detected by Western blot analysis (g) or flow cytometry analysis (h). GAPDH was used as a loading control. BEL-7402 cells were treated with either DMSO or 8.7 μ g/mL of JC for 24 hours and detected by flow cytometry analysis (i and j).
    Figure Legend Snippet: JC induces apoptosis by increasing intracellular ROS. (a, c) Effect of JC on the total ROS levels in HepG2 and BEL-7402 cells was examined by flow cytometry. HepG2 cells were treated with DMSO or 8 μ g/mL JC for 12, 24, 36, and 48 hours (a). BEL-7402 cells were treated with DMSO or 6.7, 8.7, and 10.5 μ g/mL of JC for 12 hours (c). (b) Effect of JC on superoxide anion levels in HepG2 cells was examined by flow cytometry. The levels of ROS in HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for 12, 24, 36, and 48 hours and analyzed by flow cytometry. (d–f) Effects of JC on catalase and SOD2 expression and Akt activation were determined by Western blot analysis. HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for the indicated times (d and e). BEL-7402 cells were treated with DMSO or 8.7 μ g/mL of JC for the indicated times (f). GAPDH was used as a loading control. (g–j) NAC and PEG-catalase abrogate JC-induced apoptosis. HepG2 cells were pretreated with NAC and PEG-catalase for 1 hour. HepG2 cells were treated with either DMSO or 8 μ g/mL of JC for 36 hours and detected by Western blot analysis (g) or flow cytometry analysis (h). GAPDH was used as a loading control. BEL-7402 cells were treated with either DMSO or 8.7 μ g/mL of JC for 24 hours and detected by flow cytometry analysis (i and j).

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Activation Assay, Western Blot

    20) Product Images from "SIRT2 Interacts with β-catenin to Inhibit Wnt Signaling Output in Response to Radiation-Induced Stress"

    Article Title: SIRT2 Interacts with β-catenin to Inhibit Wnt Signaling Output in Response to Radiation-Induced Stress

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-14-0223-T

    SIRT2 interacts directly with β-catenin. A, coimmunoprecipitation showing association of SIRT2 and β-catenin increases with IR. Sirt2 WT MEFs were exposed to single fractions of 2Gy, 5 Gy and 10 Gy, and compared to sham controls. Cells were harvested 24h after radiation and immunoprecipitated either with normal rabbit IgG control, SIRT2 or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. B, coimmunoprecipitation analysis showing SIRT2 associates with β-catenin in PC-3 cells. C, coimmunoprecipitation analysis showing overexpressed Flag-SIRT2 binds to β-catenin. Protein lysates from U87 glioma cells stably expressing either pcDNA3 or Flag- SIRT2 were subjected to immunoprecipitation using rabbit IgG, DDK or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. D, ROS measurement. Sirt2 WT and KO MEFs were labeled with H 2 DCFDA or carboxy DCFDA for 30 min before exposure to a single fraction of 10 Gy compared to sham controls. Cells were harvested after 30 min and analyzed. Treatment with PEG-catalase was used to scavenge ROS generated by radiation. E, Clonogenic survival assay. Sirt2 WT and KO cells were plated for clonogenic survival assay and treated with 200U/ml PEG-cat for 2h, followed by radiation. PEG-cat was left for the duration of the assay. Values represent the mean +/− SEM. *, P
    Figure Legend Snippet: SIRT2 interacts directly with β-catenin. A, coimmunoprecipitation showing association of SIRT2 and β-catenin increases with IR. Sirt2 WT MEFs were exposed to single fractions of 2Gy, 5 Gy and 10 Gy, and compared to sham controls. Cells were harvested 24h after radiation and immunoprecipitated either with normal rabbit IgG control, SIRT2 or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. B, coimmunoprecipitation analysis showing SIRT2 associates with β-catenin in PC-3 cells. C, coimmunoprecipitation analysis showing overexpressed Flag-SIRT2 binds to β-catenin. Protein lysates from U87 glioma cells stably expressing either pcDNA3 or Flag- SIRT2 were subjected to immunoprecipitation using rabbit IgG, DDK or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. D, ROS measurement. Sirt2 WT and KO MEFs were labeled with H 2 DCFDA or carboxy DCFDA for 30 min before exposure to a single fraction of 10 Gy compared to sham controls. Cells were harvested after 30 min and analyzed. Treatment with PEG-catalase was used to scavenge ROS generated by radiation. E, Clonogenic survival assay. Sirt2 WT and KO cells were plated for clonogenic survival assay and treated with 200U/ml PEG-cat for 2h, followed by radiation. PEG-cat was left for the duration of the assay. Values represent the mean +/− SEM. *, P

    Techniques Used: Immunoprecipitation, Stable Transfection, Expressing, Labeling, Generated, Clonogenic Cell Survival Assay

    21) Product Images from "Tumor Necrosis Factor–Related Apoptosis‐Inducing Ligand ( TRAIL) Promotes Angiogenesis and Ischemia‐Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide–Dependent Mechanisms"

    Article Title: Tumor Necrosis Factor–Related Apoptosis‐Inducing Ligand ( TRAIL) Promotes Angiogenesis and Ischemia‐Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide–Dependent Mechanisms

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

    doi: 10.1161/JAHA.115.002527

    TRAIL and FGF ‐2‐inducible proliferation, migration, and tubule formation involves H 2 O 2 and NO . A, TRAIL ‐inducible proliferation and (B) migration involves H 2 O 2 and NO . Growth quiescent HMEC ‐1 cells were treated with PEG ‐Catalase ( PEG ‐Cat; 200 U/mL) and l ‐ NAME (1 mmol/L) for 1 hour prior to 10 ng/mL TRAIL treatment. Cell counts were assessed 72 hours later and migration assessed 24 hours later. C, TRAIL ‐inducible tubule formation involves H 2 O 2 and NO . Serum‐starved HMEC ‐1 cells were seeded in growth factor reduced Matrigel. Cells were treated with TRAIL (10 ng/mL) and PEG ‐Cat (200 U/mL) or l ‐ NAME (1 mmol/L). Tubule formation was photographed and quantified 3 hours later. FGF ‐2‐inducible (D) proliferation, (E) migration, and (F) tubule formation involves H 2 O 2 and NO . Treatment with FGF ‐2 was identical to above except that 50 ng/mL FGF ‐2 was used. Data represent the combined results of at least 3 independent experiments. Results are expressed as mean± SEM ; 1‐way ANOVA ; * P
    Figure Legend Snippet: TRAIL and FGF ‐2‐inducible proliferation, migration, and tubule formation involves H 2 O 2 and NO . A, TRAIL ‐inducible proliferation and (B) migration involves H 2 O 2 and NO . Growth quiescent HMEC ‐1 cells were treated with PEG ‐Catalase ( PEG ‐Cat; 200 U/mL) and l ‐ NAME (1 mmol/L) for 1 hour prior to 10 ng/mL TRAIL treatment. Cell counts were assessed 72 hours later and migration assessed 24 hours later. C, TRAIL ‐inducible tubule formation involves H 2 O 2 and NO . Serum‐starved HMEC ‐1 cells were seeded in growth factor reduced Matrigel. Cells were treated with TRAIL (10 ng/mL) and PEG ‐Cat (200 U/mL) or l ‐ NAME (1 mmol/L). Tubule formation was photographed and quantified 3 hours later. FGF ‐2‐inducible (D) proliferation, (E) migration, and (F) tubule formation involves H 2 O 2 and NO . Treatment with FGF ‐2 was identical to above except that 50 ng/mL FGF ‐2 was used. Data represent the combined results of at least 3 independent experiments. Results are expressed as mean± SEM ; 1‐way ANOVA ; * P

    Techniques Used: Migration

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    Article Snippet: Reagents and antibodies N -acetyl-l -cysteine (NAC), diamide, hydrogen peroxide (H2 O2 ), MG132, PEG-catalase, tert -butyl hydroperoxide (T-hydro) and 2′–7′–dichrolodihydrofluorescein diacetate (DCF-DA) were purchased from Sigma Aldrich and Invitrogen.

    Incubation:

    Article Title: Oleoyl-Lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species
    Article Snippet: .. Cells were washed once with warm PBS, followed by incubation with 300 µL of pre-warmed assay buffer [HEPES-buffered Tyrode's solution (no FBS) containing 50 µM Amplex Red (Invitrogen) and 2 U/mL Horse Radish Peroxidase (Sigma)] containing 10 µM LPC 18:1 or PBS (vehicle) for 15 min. Polyethylene glycol catalase (PEG-catalase) 300 U/mL or polyethylene glycol superoxide dismutase (PEG-SOD) 75 U/mL (both Sigma) were added in order to detect catalase-sensitive peroxides and superoxide radicals in the medium. .. The buffer was then transferred to a black 96 well plate and fluorescence was measured at excitation and emission wavelengths of 540 and 580 nm, respectively.

    BIA-KA:

    Article Title: Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice
    Article Snippet: The probed bands were visualized by enhanced chemiluminescent substrates (ECL, Thermo Scientific) and analyzed by using Image J software (National Institutes of Health, Bethesda, MD). .. Chemicals and reagents Reagent sources were as follows: DMEM, sulindac, ET-1, PEG-catalase, PEG-SOD, apocynin (Sigma-Aldrich, St Louis, MO); DHE, C-H2 DCFDA (Invitrogen Life Technologies, Eugene, Dregon, USA); BCA protein assay kit, H2 O2 assay kit, catalase assay kit and total SOD assay kits (Beyotime, Shanghai, China). ..

    H2O2 Assay:

    Article Title: Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice
    Article Snippet: The probed bands were visualized by enhanced chemiluminescent substrates (ECL, Thermo Scientific) and analyzed by using Image J software (National Institutes of Health, Bethesda, MD). .. Chemicals and reagents Reagent sources were as follows: DMEM, sulindac, ET-1, PEG-catalase, PEG-SOD, apocynin (Sigma-Aldrich, St Louis, MO); DHE, C-H2 DCFDA (Invitrogen Life Technologies, Eugene, Dregon, USA); BCA protein assay kit, H2 O2 assay kit, catalase assay kit and total SOD assay kits (Beyotime, Shanghai, China). ..

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    Millipore peg ifn α2a
    Photomicrograph of HAK-1B cells cultured for 72 hours on a Lab-Tek Chamber slide. (A) Without <t>PEG-IFN-α2a</t> in culture medium. (B) With 4,194 ng/mL of PEG-IFN-α2a in culture medium. Apoptotic cells (short arrows) characterized by cytoplasmic shrinkage, chromatic condensation and nuclear fragmentation were noted (HE staining, X 200).
    Peg Ifn α2a, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore peg catalase
    Augmented LPS induction of TLR2 in endothelial cells requires <t>PMN</t> NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91 phox–/– mice, respectively, and were added at a concentration of 1 × 10 5 cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis ( a ) as well as Western blotting with anti-TLR2 antibody ( b ). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis ( a and b , lane 6). ( c ) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47 phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable <t>PEG-catalase</t> (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.
    Peg Catalase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Photomicrograph of HAK-1B cells cultured for 72 hours on a Lab-Tek Chamber slide. (A) Without PEG-IFN-α2a in culture medium. (B) With 4,194 ng/mL of PEG-IFN-α2a in culture medium. Apoptotic cells (short arrows) characterized by cytoplasmic shrinkage, chromatic condensation and nuclear fragmentation were noted (HE staining, X 200).

    Journal: PLoS ONE

    Article Title: Pegylated Interferon-?2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

    doi: 10.1371/journal.pone.0083195

    Figure Lengend Snippet: Photomicrograph of HAK-1B cells cultured for 72 hours on a Lab-Tek Chamber slide. (A) Without PEG-IFN-α2a in culture medium. (B) With 4,194 ng/mL of PEG-IFN-α2a in culture medium. Apoptotic cells (short arrows) characterized by cytoplasmic shrinkage, chromatic condensation and nuclear fragmentation were noted (HE staining, X 200).

    Article Snippet: Effects of PEG-IFN-α2a on the Proliferation of HCC and CHC Cell Lines in vitro The effects of PEG-IFN-α2a on the growth of the cultured cells were examined with colorimetry using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kits (Chemicon, Temecula, CA) as described elsewhere [ , ].

    Techniques: Cell Culture, Staining

    Time-course change in estimated tumor volumes of subcutaneously transplanted HAK-1B (A) or KIM-1 (B) tumors in nude mice in Experiment 1. The mice received a subcutaneous injection of 0.06 (▲), 0.6 (○), 6 (●), or 60 (∆) µg of PEG-IFN-α2a, or medium alone (Control) (□), once a week for 2 consecutive weeks. The arrows show the days of injection. The figures represent average ± SE. * P

    Journal: PLoS ONE

    Article Title: Pegylated Interferon-?2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

    doi: 10.1371/journal.pone.0083195

    Figure Lengend Snippet: Time-course change in estimated tumor volumes of subcutaneously transplanted HAK-1B (A) or KIM-1 (B) tumors in nude mice in Experiment 1. The mice received a subcutaneous injection of 0.06 (▲), 0.6 (○), 6 (●), or 60 (∆) µg of PEG-IFN-α2a, or medium alone (Control) (□), once a week for 2 consecutive weeks. The arrows show the days of injection. The figures represent average ± SE. * P

    Article Snippet: Effects of PEG-IFN-α2a on the Proliferation of HCC and CHC Cell Lines in vitro The effects of PEG-IFN-α2a on the growth of the cultured cells were examined with colorimetry using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kits (Chemicon, Temecula, CA) as described elsewhere [ , ].

    Techniques: Mouse Assay, Injection

    Photomicrograph of subcutaneous human HCC tumor in nude mice that was developed after the injection of HAK-1B cells. (A) A control mouse that received culture medium alone. The tumor shows a compact arrangement of tumor cells and a sinusoid-like structure in the stroma. (B) A mouse that received a s.c. injection of 0.06 μg of PEG-IFN-α2a. There are some apoptotic tumor-cells characterized by shrinkage and eosinophilic change in the cytoplasm, chromatin condensation and/or fragmentation of nuclei (arrows, HE staining, X200). (C) The same tumor as shown in (B). There are some TUNEL-positive cells showing brown nuclei (arrows, stained by the TUNEL technique, X200).

    Journal: PLoS ONE

    Article Title: Pegylated Interferon-?2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

    doi: 10.1371/journal.pone.0083195

    Figure Lengend Snippet: Photomicrograph of subcutaneous human HCC tumor in nude mice that was developed after the injection of HAK-1B cells. (A) A control mouse that received culture medium alone. The tumor shows a compact arrangement of tumor cells and a sinusoid-like structure in the stroma. (B) A mouse that received a s.c. injection of 0.06 μg of PEG-IFN-α2a. There are some apoptotic tumor-cells characterized by shrinkage and eosinophilic change in the cytoplasm, chromatin condensation and/or fragmentation of nuclei (arrows, HE staining, X200). (C) The same tumor as shown in (B). There are some TUNEL-positive cells showing brown nuclei (arrows, stained by the TUNEL technique, X200).

    Article Snippet: Effects of PEG-IFN-α2a on the Proliferation of HCC and CHC Cell Lines in vitro The effects of PEG-IFN-α2a on the growth of the cultured cells were examined with colorimetry using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kits (Chemicon, Temecula, CA) as described elsewhere [ , ].

    Techniques: Mouse Assay, Injection, Staining, TUNEL Assay

    Anti-proliferative effect of PEG-IFN-α2a. (A) Chronological changes in relative viable cell number (% of the control) after adding 4,194 ng/mL of PEG-IFN-α2a. Growth was suppressed with time in 8 cell lines. (B) 96 hours after adding 10 different concentrations of PEG-IFN-α2a. Cell proliferation was suppressed in a dose-dependent manner in 11 cell lines. The suppression was significant ( P

    Journal: PLoS ONE

    Article Title: Pegylated Interferon-?2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

    doi: 10.1371/journal.pone.0083195

    Figure Lengend Snippet: Anti-proliferative effect of PEG-IFN-α2a. (A) Chronological changes in relative viable cell number (% of the control) after adding 4,194 ng/mL of PEG-IFN-α2a. Growth was suppressed with time in 8 cell lines. (B) 96 hours after adding 10 different concentrations of PEG-IFN-α2a. Cell proliferation was suppressed in a dose-dependent manner in 11 cell lines. The suppression was significant ( P

    Article Snippet: Effects of PEG-IFN-α2a on the Proliferation of HCC and CHC Cell Lines in vitro The effects of PEG-IFN-α2a on the growth of the cultured cells were examined with colorimetry using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kits (Chemicon, Temecula, CA) as described elsewhere [ , ].

    Techniques:

    Augmented LPS induction of TLR2 in endothelial cells requires PMN NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91 phox–/– mice, respectively, and were added at a concentration of 1 × 10 5 cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis ( a ) as well as Western blotting with anti-TLR2 antibody ( b ). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis ( a and b , lane 6). ( c ) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47 phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable PEG-catalase (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.

    Journal: Journal of Clinical Investigation

    Article Title: TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase

    doi: 10.1172/JCI200318696

    Figure Lengend Snippet: Augmented LPS induction of TLR2 in endothelial cells requires PMN NADPH oxidase and PMN adhesion. Studies determined the effects of PMNs cocultured with MLVECs on MLVEC TLR2 expression in response to LPS. MLVECs were isolated and cultured as described in Methods and treated with LPS (1 μg/ml) for 2 hours. The cocultured PMNs were isolated from WT and gp91 phox–/– mice, respectively, and were added at a concentration of 1 × 10 5 cells/ml. At the end of incubation with LPS, MLVECs were washed with HBSS three times and then lysed with lysis buffer. Cell lysates were subjected to extraction of total RNA and RT-PCR analysis ( a ) as well as Western blotting with anti-TLR2 antibody ( b ). To address the effects of CD18 on PMN-activated TLR2 expression in MLVECs, confluent MLVECs were treated with LPS in the presence of WT PMNs and anti-CD18 antibody for 2 hours, followed by washing with HBSS three times. This was followed by RT-PCR and Western analysis ( a and b , lane 6). ( c ) To address the role of endogenous endothelial NADPH oxidase in the regulation of TLR2 expression, MLVECs isolated from p47 phox–/– mice were stimulated with LPS and cocultured with or without WT PMNs for the times indicated. To address the role of oxidants derived from PMNs in LPS-induced TLR2 expression in endothelial cells, membrane-permeable PEG-catalase (1,000 U/ml) was applied to the coculture system. The data are representative of three independent studies.

    Article Snippet: In some experiments, to further address the role of PMN-derived oxidants in the mechanism of LPS-induced TLR2 upregulation in endothelial cells, PEG-catalase (1,000 U/ml, Sigma-Aldrich) was added to the coculture system.

    Techniques: Expressing, Isolation, Cell Culture, Mouse Assay, Concentration Assay, Incubation, Lysis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay

    LPC 18:1 induces intracellular and extracellular superoxide production. (A) ROS levels were measured as in Figure 2 after inhibition of SOD with 20 µM DETCA. (B) Cells plated in 96-well dishes were incubated with 15 µM DHE in the presence of 20 µM DETCA at 37°C for 15 min. Thereafter, cells were exposed to 60 µM LPC 18:1 or PBS (vehicle) in PBS containing 5% FBS at 37°C for 15 min, followed by fluorometric superoxide quantification. (C) Cells grown on a glass coverslip were treated as in B followed by fluorescent microscopy. (D) Amplex Red Assay was performed in cells exposed to 10 µM LPC 18:1 or PBS in the presence or absence of PEG-catalase (300 U/ml) or PEG-SOD (75 U/ml) in HEPES-buffered Tyrode's solution without FBS at 37°C for 15 min. The presented values are catalase-sensitive values obtained by subtraction of values obtained in the presence of catalase from those in the absence of catalase. The values shown are mean ± SEM of 3 independent experiments performed in triplicates and analyzed by one-way ANOVA with Tukey's post-hoc test (A,D) or unpaired t-test (B,C). If otherwise not indicated, asterisks show significance compared to PBS control; *p

    Journal: PLoS ONE

    Article Title: Oleoyl-Lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species

    doi: 10.1371/journal.pone.0113443

    Figure Lengend Snippet: LPC 18:1 induces intracellular and extracellular superoxide production. (A) ROS levels were measured as in Figure 2 after inhibition of SOD with 20 µM DETCA. (B) Cells plated in 96-well dishes were incubated with 15 µM DHE in the presence of 20 µM DETCA at 37°C for 15 min. Thereafter, cells were exposed to 60 µM LPC 18:1 or PBS (vehicle) in PBS containing 5% FBS at 37°C for 15 min, followed by fluorometric superoxide quantification. (C) Cells grown on a glass coverslip were treated as in B followed by fluorescent microscopy. (D) Amplex Red Assay was performed in cells exposed to 10 µM LPC 18:1 or PBS in the presence or absence of PEG-catalase (300 U/ml) or PEG-SOD (75 U/ml) in HEPES-buffered Tyrode's solution without FBS at 37°C for 15 min. The presented values are catalase-sensitive values obtained by subtraction of values obtained in the presence of catalase from those in the absence of catalase. The values shown are mean ± SEM of 3 independent experiments performed in triplicates and analyzed by one-way ANOVA with Tukey's post-hoc test (A,D) or unpaired t-test (B,C). If otherwise not indicated, asterisks show significance compared to PBS control; *p

    Article Snippet: Cells were washed once with warm PBS, followed by incubation with 300 µL of pre-warmed assay buffer [HEPES-buffered Tyrode's solution (no FBS) containing 50 µM Amplex Red (Invitrogen) and 2 U/mL Horse Radish Peroxidase (Sigma)] containing 10 µM LPC 18:1 or PBS (vehicle) for 15 min. Polyethylene glycol catalase (PEG-catalase) 300 U/mL or polyethylene glycol superoxide dismutase (PEG-SOD) 75 U/mL (both Sigma) were added in order to detect catalase-sensitive peroxides and superoxide radicals in the medium.

    Techniques: Inhibition, Incubation, Microscopy, Amplex Red Assay

    VL are enriched in and functionally dependent on NADPH oxidase activity. (a) MVECs coexpressing p47phox-DsRed and actin-GFP were imaged live during T cell diapedesis. p47phox was enriched in ventral (IRM) actin-rich initiation nodes (N) and in VL leading edge (arrowheads). See Video 10 . (b) Representative ratiometric images of H 2 O 2 production in (i) pore- and (ii) gap-closing VL in MVECs expressing the biosensor mHyPer during T cell diapedesis. (c) Quantification of p47phox (i) and H 2 O 2 (ii) enrichment in VL. n > 13. (d, i) Representative NADPH oxidase inhibition/washout imaging experiment. After multiple T cell diapedesis events on MVECs expressing mYFP, apocynin was added. Arrows indicate multiple pores and gaps that persisted for 15 min, but were rapidly healed by steered VL (arrowheads) after drug washout. See Video 10. Experiments as in d (i) were performed with VAS-2870, DPI, PEG-catalase, and DMSO (control) and quantified by cumulative wound closure analysis, shown as a continuous line plot (ii) and bar graph of single pre/post-washout time points (iii). n > 5. (e) MVECs coexpressing mDsRed and mHyPer were imaged during probe wounds and after addition of VAS-2870. Representative mDsRed images (i), kymograph (ii), mHyper ratiometic images (iii), and quantitation (iv) are shown. n = 3. Values represent mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P

    Journal: The Journal of Cell Biology

    Article Title: Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds

    doi: 10.1083/jcb.201209077

    Figure Lengend Snippet: VL are enriched in and functionally dependent on NADPH oxidase activity. (a) MVECs coexpressing p47phox-DsRed and actin-GFP were imaged live during T cell diapedesis. p47phox was enriched in ventral (IRM) actin-rich initiation nodes (N) and in VL leading edge (arrowheads). See Video 10 . (b) Representative ratiometric images of H 2 O 2 production in (i) pore- and (ii) gap-closing VL in MVECs expressing the biosensor mHyPer during T cell diapedesis. (c) Quantification of p47phox (i) and H 2 O 2 (ii) enrichment in VL. n > 13. (d, i) Representative NADPH oxidase inhibition/washout imaging experiment. After multiple T cell diapedesis events on MVECs expressing mYFP, apocynin was added. Arrows indicate multiple pores and gaps that persisted for 15 min, but were rapidly healed by steered VL (arrowheads) after drug washout. See Video 10. Experiments as in d (i) were performed with VAS-2870, DPI, PEG-catalase, and DMSO (control) and quantified by cumulative wound closure analysis, shown as a continuous line plot (ii) and bar graph of single pre/post-washout time points (iii). n > 5. (e) MVECs coexpressing mDsRed and mHyPer were imaged during probe wounds and after addition of VAS-2870. Representative mDsRed images (i), kymograph (ii), mHyper ratiometic images (iii), and quantitation (iv) are shown. n = 3. Values represent mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P

    Article Snippet: Inhibitors of NADPH oxidase signaling used included VAS-2870 (Nox2/4 inhibitor; 15 µM; Enzo Life Sciences), DPI (non-specific Nox inhibitor/Flavoprotein inhibitor; 12.5 µM; Sigma-Aldrich), apocynin (inhibitor of p47phox assembly into Nox complex/H2 O2 scavenger; 1 mM; Sigma-Aldrich), PEG-catalase (H2 O2 degrading enzyme; 200 U/ml; Sigma-Aldrich), and Tempol (superoxide dismutase/catalase mimetic; 500 µM; Sigma-Aldrich).

    Techniques: Activity Assay, Expressing, Inhibition, Imaging, Quantitation Assay