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  • 99
    Millipore nadph
    Role of gpr in maintenance of redox homeostasis of M. smegmatis. (A,B) Levels of oxidized <t>(NADP)</t> form of nicotinamide adenine dinucleotide phosphate in biofilms of wild-type, Δ gpr and the complemented strains cultured in normal Sauton’s medium. (C) Ratio of <t>NADPH</t> to NADP calculated from (A,B) . Data represent mean of three biologically independent experiments ∗ , ∗∗ , and ∗∗∗ indicate p ( t -test)
    Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nox4  (Abcam)
    99
    Abcam nox4
    GKT137831 attenuates TGFβ1‐mediated fibroblast activation. Primary prostate human fibroblasts were treated with bFGF or TGFβ1 in the presence of 30 μM GKT137831 (GKT) or DMSO equivalent (control, ctrl) for 48 hr before ( a ) qPCR of Nox1 or <t>Nox4</t> expression relative to the housekeeping gene TBP, ( b ) Western blotting of total cell extracts using the indicated antibodies and GAPDH as loading control, ( c ) determination of extracellular H 2 O 2 levels via Amplex Red assay, ( d ) quantification of intracellular ROS via CM‐H 2 DCFDA staining, ( e ) qPCR determination of the indicated CAF markers, and ( f , g ) analysis of migration using Boyden chamber transwell assays. ( a , c – e , g ) Data represent mean + SEM of at least four independent experiments using primary fibroblasts isolated from different donors. Statistical significance is shown (** p
    Nox4, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β nadph
    NOS detection in chromatophore organs of S. officinalis . A–C , <t>NADPH</t> diaphorase reaction. A , embryo chromatophore organs. The NADPH diaphorase staining is visible in the cytoelastic sacculus, which is not fully pigmented. B , isolated juvenile chromatophore organ showing the staining in the fully pigmented sacculus and in the relaxed and contracted muscle fibers. C , isolated adult chromatophore organ with the reaction localized in contracted muscle fibers. D–F , controls of embryo, juvenile, and adult chromatophore organs with omission of <t>β-NADPH</t> showing loss of reaction and solubilization of embryo and juvenile chromatophore pigment ( D and E ). G–I , immunohistochemistry/cytochemistry reaction. G , embryo chromatophore organs. The immunopositivity is localized in the cytoelastic sacculus. H , isolated juvenile chromatophore organ showing the immunoreactivity in the pigment sacculus and in the contracted and partially relaxed muscle fibers. I , Sepia isolated adult chromatophore organ. The immunopositivity is visible in contracted and partially relaxed muscle fibers. Scale bars , 20 μm. mf , muscle fiber; sa , sacculus.
    β Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nicotinamide adenine dinucleotide phosphate nadph
    Increased HO-1 expression leads to inhibition of <t>PDGF-stimulated</t> <t>NADPH</t> oxidase activity
    Nicotinamide Adenine Dinucleotide Phosphate Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Ushio m localizing nadph oxidase derived ros
    Anti-angiogenic therapy targeting <t>NADPH</t> oxidase <t>(ROS)-dependent</t> VEGF signaling
    M Localizing Nadph Oxidase Derived Ros, supplied by Ushio, used in various techniques. Bioz Stars score: 89/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore m nadp
    GAGs from paw/ankle or knee/elbow were partial inhibitors of enzymatic activity of <t>GPI.</t> Each well contained 4.2 units/ml glucose-6-phosphate dehydrogenase, 1.0 m m <t>NADP,</t> 0.4 μg/ml (6.3 μ m ) recombinant mouse GPI, and inhibitors at various
    M Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam nadp nadph assay kit
    Transcription factors that determine B cell identity regulate activity of the pentose phosphate pathway and dependency on PP2A (A) Human embryonic kidney cells (HEK-293) were transduced with a polycistronic doxycycline (Dox)-inducible vector to express the B-lineage transcription factors Pax5, Ikaros, Ebf1 and Tcf3, as confirmed by Western blot after Dox treatment for the times indicated. (B) Relative <t>NADPH/NADP</t> ratios were measured before and after 24 hours of Dox-induction of the polycistronic vector (Pax5, Ikaros, Ebf1 and Tcf3) or EV. (C) Murine myeloid leukemia cells were transduced with the inducible polycistronic vector for myeloid→B cell reprogramming. Phenotypic changes were monitored by flow cytometry 48 hours after induction of Dox and expression of Pax5, Ikaros, Ebf1 and Tcf3 was confirmed by Western blot. (E) In the same cells, protein levels for PP2A Sub A, PP2A Sub C and G6pdx were measured by Western blot. (F) Human small cell lung cancer (SCLC) and normal lung epithelial (BEAS2B) cell lines were used to compare PAX5, PP2A and G6PD expression by Western blot. (G) NADPH/NADP ratios were measured in PAX5 + and PAX5 − SLCL cell lines and upon expression of PAX5 or EV. (H) Murine Pax5 +/− Ebf1 +/− BCR-ABL1 B-ALL cells were treated with the PP2A-inhibitor LB-100 at the concentrations indicated. Dose response curves depicting relative cell viability after 72 hours are depicted. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.
    Nadp Nadph Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson nadph regenerating system
    Extract ion ( m / z 956 → 817) chromatograms obtained from LC-LTQ MS analysis of microsomal incubations containing LIM, GSH, NAL, and <t>NADPH</t> in the absence microsomes (a), or in presence of HLMs (b) or <t>MLMs</t> (c). (d) Extracted ion ( m / z 956 → 817) chromatogram obtained from LC-LTQ MS analysis of synthetic M1 and M1′. (e) MS/MS spectrum of M1 generated in microsomal incubations (M1′ showed the same MS/MS spectrum). (f) MS/MS spectrum of synthetic M1 (synthetic M1′ showed the same MS/MS spectrum).
    Nadph Regenerating System, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision nadp nadph quantification kit
    Redox homeostasis in SKOV3/DDP cells is maintained intrinsically by pairing oxidative phosphorylation with pentose phosphate pathway. (A) Cellular <t>NADPH</t> content and (B) <t>NADPH/NADP</t> + ratio, (C) GSH and (D) GSSG contents, (E) total GSH (GSH plus GSSG) level and (F) GSH/GSSG ratio determined using enzymatic assays. (G) The expression level G6PD gene was detected using reverse transcription-quantitative polymerase chain reaction. (H) G6PD protein expression level was determined using western blotting, and the enzymatic activity was analyzed using a G6PD assay kit. The data are representative of three experiments. (I) Cell viability of SKOV3 or SKOV3/DDP cells was determined using a MTT assay in the presence of G6PD inhibitors (20 μ M 6-AN or 250 μ M DHEA) with or without cisplatin for 24 h. * P
    Nadp Nadph Quantification Kit, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore nadp nadph quantification kit
    Nutlin inhibits or promotes PPP. ( A ) Cells were treated with vehicle or Nutlin (10 μM) and/or MK2206 (10 μM) for 24 h. Lysates were analyzed for <t>NADPH.</t> Average NADPH level from triplicate was presented with SD indicated. ( B ) Cells were transfected with control siRNA, TIGAR siRNA, or G6PD siRNA and then treated with vehicle or Nutlin (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated (left panel). Lysates were also immunoblotted for the indicated proteins (right panel). ( C ) cells were treated with Nutlin for a 12-h period and then fluxed with D-[1,2- 13 C] glucose for 15 min. Metabolites were analyzed by LC–MS. Ribose-5-P- 13 C1 levels in vehicle-treated and Nutlin-treated cells were presented with SD indicated. There is no significant difference between vehicle and Nutlin in U2OS cells ( P = 0.22). There is significant difference between vehicle and Nutlin in MHM cells ( P = 0.008). ( D ) Metabolism of 1,2- 13 C 2 -glucose through PPP to generate NADPH and into ribose-5-P- 13 C1 is schematically presented.
    Nadp Nadph Quantification Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Applichem nadph
    MJ25 is an inhibitor of thioredoxin reductase 1 (TrxR1) a. The capability of MJ25 and auranofin to inhibit recombinant, rat-derived TrxR1 in vitro was measured by an <t>NADPH</t> dependent 5,5′-dithiobis-[2-nitrobenzoic acid] (DTNB) assay. b. ARN8 cells were treated with MJ25, auranofin or DMSO, respectively, for the indicated periods of time. TrxR1 inhibition was subsequently assessed in cell lysates by an NADPH and Trx dependent insulin reduction endpoint assay, measuring thiol formation using DTNB. Ratios between MJ25 and DMSO as well as auranofin and DMSO were determined for each point in time. (c and d) ARN8 cells were treated with c. MJ25 or d. auranofin, while in each half of the samples growth media were supplemented with sodium selenite [75 nM] three days prior to seeding as well as during seeding and treatment for 72 hours. Cell viability and clonogenic capacity were determined. e. ROS levels were determined in ARN8 cells 3 hours after the indicated treatment by measuring fluorescence of 2′,7′-dichlorofluorescein (DCF). f. Induction of anti-oxidative proteins by MJ25 and auranofin was investigated in ARN8 cells at the indicated points in time by Western blotting. DMSO served as vehicle control (0 μM). g. ARN8 cells were pre-treated with L-buthionine sulfoximine (BSO) or vehicle (H 2 O) for 72 hours, upon which cells were re-plated in BSO- and vehicle-free growth medium. Cell viability was assessed by SRB assay after 72 hours in the presence of vehicle (DMSO), MJ25 (left panel) or auranofin (middle panel), respectively. Intracellular glutathione (GSH) levels were determined 72 hours after BSO / vehicle treatment (right panel). h. Inhibition of yeast-derived glutathione reductase by MJ25 and auranofin was determined in vitro by measurement of glutathione disulfide (GSSG) reduction.
    Nadph, supplied by Applichem, used in various techniques. Bioz Stars score: 93/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nadp  (Abcam)
    99
    Abcam nadp
    Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced <t>NADP</t> and <t>NAD</t> ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P
    Nadp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega nicotinamide adenine dinucleotide cell based assays
    The central carbon metabolism (CCM). The CCM combines enzymatic reactions that convert carbon sources into biomass precursors. This figure shows the five main pathways forming the CCM: glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle (TCA), lipogenesis, and beta-oxidation. Two opposite metabolic demands are at the core of the CCM: anabolic reactions, which consist in biomass synthesis, and catabolic reactions, leading to the breakdown of macromolecules for energetic use. These two aspects of <t>cell</t> metabolism are managed by biochemical oscillators, including redox couples, such as <t>nicotinamide</t> <t>adenine</t> <t>dinucleotide</t> (NAD + /NADH) and nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD + /NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP + /NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP + /NADPH ratio triggers lipogenesis. The ATP/(ADP + Pi) ratio senses the metabolic state of the cell and may lead to metabolic switches in the CCM.
    Nicotinamide Adenine Dinucleotide Cell Based Assays, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt hydrate nadph
    Activity of thioredoxin (Trx) system enzymes as measured by the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and insulin reduction assay. ( a ) Recovery of selenoenzyme thioredoxin reductase (TrxR, 20 nM) activity inhibited by methylmercury (MeHg, 200 nM) by the chelating agents [15]aneN 4 S, BAL and DMSA (40 µM) as measured by the DTNB reduction assay. ( b ) Recovery of Trx system activity by chelating agents ([15]aneN 4 S, BAL and DMSA; 40 µM) following incubation of TrxR (20 nM), Trx (3 µM) and <t>β-nicotinamide</t> adenine dinucleotide 2′-phosphate reduced <t>tetrasodium</t> salt hydrate <t>(NADPH)</t> with MeHg (200 nM) as measured by the insulin reduction assay. All data points represent the mean ± SE of at least three independent experiments. * Different from control, p
    β Nicotinamide Adenine Dinucleotide 2 Phosphate Reduced Tetrasodium Salt Hydrate Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH nadph
    Physiological aspects of antimicrobial activity by HD-6. ( a ) Oxidized HD-6 (top row) or truncated ΔoxHD-6 (bottom row) were incubated with rat thioredoxin reductase, <t>NADPH,</t> and with or without 3 μ M thioredoxin (TRX). Enzymatic conversion from oxidized HD-6 into the reduced form was investigated by RP-HPLC analysis. Representative experiments of three independent experiments are shown. ( b ) Schematic model describing a potential mechanism of HD-6 activity. HD-6 might be secreted as an oxidized peptide by Paneth cells into the crypt, trap bacteria by formation of nanonets that further diffuse toward the lumen. The reducing milieu might reduce HD-6 that eventually kills bacteria. For more details please see text. ( c ) Bif. adolescentis Ni3,29c bacteria were incubated in the absence (untreated) or presence of oxidized HD-6 for 30 min without anaerobic environment, followed by 90 min of incubation in an anaerobic environment in the presence or absence of DTT. Aliquots were plated on agar plates and colony-forming units were calculated the next day. Experiments were carried out at least three times; means with s.e.m. are shown. DTT, dithiothreitol; HD-6, α-defensin 6; <t>oxHD-6,</t> oxidized HD-6; redHD-6, reduced HD-6; RP-HPLC, reversed-phase high-performance liquid chromatography; UV, ultraviolet.
    Nadph, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Journal of Biological Chemistry nadph oxidase 5
    Physiological aspects of antimicrobial activity by HD-6. ( a ) Oxidized HD-6 (top row) or truncated ΔoxHD-6 (bottom row) were incubated with rat thioredoxin reductase, <t>NADPH,</t> and with or without 3 μ M thioredoxin (TRX). Enzymatic conversion from oxidized HD-6 into the reduced form was investigated by RP-HPLC analysis. Representative experiments of three independent experiments are shown. ( b ) Schematic model describing a potential mechanism of HD-6 activity. HD-6 might be secreted as an oxidized peptide by Paneth cells into the crypt, trap bacteria by formation of nanonets that further diffuse toward the lumen. The reducing milieu might reduce HD-6 that eventually kills bacteria. For more details please see text. ( c ) Bif. adolescentis Ni3,29c bacteria were incubated in the absence (untreated) or presence of oxidized HD-6 for 30 min without anaerobic environment, followed by 90 min of incubation in an anaerobic environment in the presence or absence of DTT. Aliquots were plated on agar plates and colony-forming units were calculated the next day. Experiments were carried out at least three times; means with s.e.m. are shown. DTT, dithiothreitol; HD-6, α-defensin 6; <t>oxHD-6,</t> oxidized HD-6; redHD-6, reduced HD-6; RP-HPLC, reversed-phase high-performance liquid chromatography; UV, ultraviolet.
    Nadph Oxidase 5, supplied by Journal of Biological Chemistry, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti nox4
    Effect of DBTG on expression of <t>NOX4</t> in BLM-induced rats PF. (a) Representative western-blots of NOX4 and β -actin at 7 days ( n = 3); (b) representative western-blots of NOX4 and β -actin at 14 days ( n = 3); (c) representative western-blots of NOX4 and β -actin at 28 days ( n = 3). The expression of NOX4 was inhibited in DBTG treatment groups contrast with model group. Data were expressed as mean ± SEM, ∗ p
    Anti Nox4, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega nadph regenerating system
    Metabolism of nicotine by recombinantly expressed aphid (CYP6CY3) and human (CYP2A6) P450s. ( A ) The recovery of nicotine after 10 μM nicotine was incubated with 50 pmol P450+reductase in the presence or absence of <t>NADPH</t> for 60 min. Mean data ±
    Nadph Regenerating System, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Carl Roth GmbH nadp
    Overall structure model ( A ) of ecQueF with preQ 0 and <t>NADPH</t> and close-up view of Trp 57 ( B ) and Trp 198 ( C ). Tryptophan is shown in magenta . Other amino acids in the active site and NADPH are shown in blue and yellow , respectively. Cys 190 , preQ 0 , and amino acids having hydrophilic interaction with Trp 198 .
    Nadp, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Melford Laboratories nadp
    (A and B) MS analysis of the alcohol product from an incubation of 4-pyridine carboxaldehyde, enzyme, and either NADPH (A) or (4 R )-[4- 2 <t>H]NADPD</t> (B). The corresponding GC traces can be seen in Fig. S2 in the supplemental material. (C) NMR spectra showing the purified nucleotide reaction product obtained from incubations of Cbei_3974 with (4 R )-[4- 2 H]NADPD and 4-pyridine carboxaldehyde (red) against a standard of <t>NADP</t> + (blue).
    Nadp, supplied by Melford Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nadph oxidase inhibitor apocynin
    Involvement of ROS in leptin-mediated GATA-4 nuclear translocation. Effect of the <t>NADPH</t> oxidase inhibitor <t>apocynin</t> (1 μmol/L) on leptin-induced GATA-4 nuclear translocation in RASMC. GATA-4 antibody was visualized by CruzFluor 488-conjugated secondary antibody (second panel; green) while Acti-stain 555 phalloidin was used to stain F-actin (third panel; red). DAPI stained the nuclei blue (first panel; blue). The overlay of DAPI, GATA-4, and F-actin (merged) is shown in the right panel. ROS depletion inhibited GATA-4 nuclear translocation.
    Nadph Oxidase Inhibitor Apocynin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Role of gpr in maintenance of redox homeostasis of M. smegmatis. (A,B) Levels of oxidized (NADP) form of nicotinamide adenine dinucleotide phosphate in biofilms of wild-type, Δ gpr and the complemented strains cultured in normal Sauton’s medium. (C) Ratio of NADPH to NADP calculated from (A,B) . Data represent mean of three biologically independent experiments ∗ , ∗∗ , and ∗∗∗ indicate p ( t -test)

    Journal: Frontiers in Microbiology

    Article Title: GlnR Activation Induces Peroxide Resistance in Mycobacterial Biofilms

    doi: 10.3389/fmicb.2018.01428

    Figure Lengend Snippet: Role of gpr in maintenance of redox homeostasis of M. smegmatis. (A,B) Levels of oxidized (NADP) form of nicotinamide adenine dinucleotide phosphate in biofilms of wild-type, Δ gpr and the complemented strains cultured in normal Sauton’s medium. (C) Ratio of NADPH to NADP calculated from (A,B) . Data represent mean of three biologically independent experiments ∗ , ∗∗ , and ∗∗∗ indicate p ( t -test)

    Article Snippet: Purified NADP (Sigma # 10128031001) and NADPH (Sigma # 10107824001) were used for standard curves, which were used for determination of the nucleotides in each sample.

    Techniques: Cell Culture

    GKT137831 attenuates TGFβ1‐mediated fibroblast activation. Primary prostate human fibroblasts were treated with bFGF or TGFβ1 in the presence of 30 μM GKT137831 (GKT) or DMSO equivalent (control, ctrl) for 48 hr before ( a ) qPCR of Nox1 or Nox4 expression relative to the housekeeping gene TBP, ( b ) Western blotting of total cell extracts using the indicated antibodies and GAPDH as loading control, ( c ) determination of extracellular H 2 O 2 levels via Amplex Red assay, ( d ) quantification of intracellular ROS via CM‐H 2 DCFDA staining, ( e ) qPCR determination of the indicated CAF markers, and ( f , g ) analysis of migration using Boyden chamber transwell assays. ( a , c – e , g ) Data represent mean + SEM of at least four independent experiments using primary fibroblasts isolated from different donors. Statistical significance is shown (** p

    Journal: International Journal of Cancer

    Article Title: Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

    doi: 10.1002/ijc.31316

    Figure Lengend Snippet: GKT137831 attenuates TGFβ1‐mediated fibroblast activation. Primary prostate human fibroblasts were treated with bFGF or TGFβ1 in the presence of 30 μM GKT137831 (GKT) or DMSO equivalent (control, ctrl) for 48 hr before ( a ) qPCR of Nox1 or Nox4 expression relative to the housekeeping gene TBP, ( b ) Western blotting of total cell extracts using the indicated antibodies and GAPDH as loading control, ( c ) determination of extracellular H 2 O 2 levels via Amplex Red assay, ( d ) quantification of intracellular ROS via CM‐H 2 DCFDA staining, ( e ) qPCR determination of the indicated CAF markers, and ( f , g ) analysis of migration using Boyden chamber transwell assays. ( a , c – e , g ) Data represent mean + SEM of at least four independent experiments using primary fibroblasts isolated from different donors. Statistical significance is shown (** p

    Article Snippet: Consistent with our previous findings that Nox4 is strongly upregulated in activated fibroblasts, significantly higher numbers of Nox4 mRNA‐expressing cells (0.73%, p < 0.001) and an abundance of densely stained clusters (denoting the presence of multiple transcripts/cell) were observed in PCa, particularly within the peritumoral stroma (Supporting Information, Fig. S2).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Amplex Red Assay, Staining, Migration, Isolation

    Nox4 but not Nox1 is critical for TGFβ1‐mediated fibroblast activation. ( a ) Primary human prostate fibroblasts incubated with bFGF or TGFβ1 as indicated for 48 hr before qPCR analysis of Nox family members relative to the housekeeping gene TBP. The direct TGFβ1 target genes SERPINE1 and COMP served as positive controls. The nonresponsive gene HMBS served as negative control. ( b ) Lentiviral‐mediated silencing of Nox1 or Nox4 in primary human prostate fibroblasts transduced with 2 different Nox1 or Nox4‐specific shRNAs (1 and 2) and subsequently treated with bFGF or TGFβ1 for 48 hr before qPCR of Nox1 and Nox4 expression ( top) , or the indicated CAF marker ( bottom) relative to the housekeeping gene TBP. ( c , d ) Nox1 RNA in situ hybridization on radical prostatectomy prostate tissue specimens from PCa patients. Benign (BE) and adjacent cancer (CA) areas are shown. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Boxed regions ( top panel ) are enlarged ( lower panel ). Arrowheads demarcate weak epithelial Nox1 mRNA levels in benign tissue. ( d ) ImageJ quantification of Nox1 in situ hybridization images as in ( c ) using threshold settings described in Materials and Methods. ( a , b ) Data represent mean + SEM of four independent experiments using primary fibroblasts isolated from different donors. ( c ) Images are representative of 3 independent experiments using tissue from different patients. ( d ) Data represent mean + SEM from 10 fields of view each (40× magnification) from CA and BE adjacent regions of 3 different patient specimens. ( a , b , d ) Statistical significance is shown (* p

    Journal: International Journal of Cancer

    Article Title: Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

    doi: 10.1002/ijc.31316

    Figure Lengend Snippet: Nox4 but not Nox1 is critical for TGFβ1‐mediated fibroblast activation. ( a ) Primary human prostate fibroblasts incubated with bFGF or TGFβ1 as indicated for 48 hr before qPCR analysis of Nox family members relative to the housekeeping gene TBP. The direct TGFβ1 target genes SERPINE1 and COMP served as positive controls. The nonresponsive gene HMBS served as negative control. ( b ) Lentiviral‐mediated silencing of Nox1 or Nox4 in primary human prostate fibroblasts transduced with 2 different Nox1 or Nox4‐specific shRNAs (1 and 2) and subsequently treated with bFGF or TGFβ1 for 48 hr before qPCR of Nox1 and Nox4 expression ( top) , or the indicated CAF marker ( bottom) relative to the housekeeping gene TBP. ( c , d ) Nox1 RNA in situ hybridization on radical prostatectomy prostate tissue specimens from PCa patients. Benign (BE) and adjacent cancer (CA) areas are shown. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Boxed regions ( top panel ) are enlarged ( lower panel ). Arrowheads demarcate weak epithelial Nox1 mRNA levels in benign tissue. ( d ) ImageJ quantification of Nox1 in situ hybridization images as in ( c ) using threshold settings described in Materials and Methods. ( a , b ) Data represent mean + SEM of four independent experiments using primary fibroblasts isolated from different donors. ( c ) Images are representative of 3 independent experiments using tissue from different patients. ( d ) Data represent mean + SEM from 10 fields of view each (40× magnification) from CA and BE adjacent regions of 3 different patient specimens. ( a , b , d ) Statistical significance is shown (* p

    Article Snippet: Consistent with our previous findings that Nox4 is strongly upregulated in activated fibroblasts, significantly higher numbers of Nox4 mRNA‐expressing cells (0.73%, p < 0.001) and an abundance of densely stained clusters (denoting the presence of multiple transcripts/cell) were observed in PCa, particularly within the peritumoral stroma (Supporting Information, Fig. S2).

    Techniques: Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Negative Control, Transduction, Expressing, Marker, RNA In Situ Hybridization, In Situ Hybridization, Isolation

    Nox4 expression is elevated in clinical PCa. PCa TMA stained via ISH for Nox4 transcript. ( a ) Representative images of benign (BE) or cancer (CA) foci are shown. Low‐grade and high‐grade CA was defined as predominantly Gleason pattern 3 or lower (Gleason score ≤3 + 4) and predominantly Gleason pattern 4 or higher (Gleason score ≥4 + 3), respectively. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Boxed regions ( top panel ) are enlarged ( lower panel ). Arrowheads demarcate weak Nox4 staining in benign tissue. Original magnification: 20× ( top panel ) and 40× ( lower panel ). ( b – d ) Quick‐score quantification of ( a ) as described in Materials and Methods. ( b ) Comparison of benign (BE) versus cancer (CA), ( c ) stratification of cancer cases into low or high Gleason grading as defined in ( a ) or ( d ) stratification of cancer cases according to ERG ‐fusion status. ( b – d ) Data represent mean + SEM. Number of patients analyzed is indicated ( n ). Statistical significance is shown (* p

    Journal: International Journal of Cancer

    Article Title: Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

    doi: 10.1002/ijc.31316

    Figure Lengend Snippet: Nox4 expression is elevated in clinical PCa. PCa TMA stained via ISH for Nox4 transcript. ( a ) Representative images of benign (BE) or cancer (CA) foci are shown. Low‐grade and high‐grade CA was defined as predominantly Gleason pattern 3 or lower (Gleason score ≤3 + 4) and predominantly Gleason pattern 4 or higher (Gleason score ≥4 + 3), respectively. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Boxed regions ( top panel ) are enlarged ( lower panel ). Arrowheads demarcate weak Nox4 staining in benign tissue. Original magnification: 20× ( top panel ) and 40× ( lower panel ). ( b – d ) Quick‐score quantification of ( a ) as described in Materials and Methods. ( b ) Comparison of benign (BE) versus cancer (CA), ( c ) stratification of cancer cases into low or high Gleason grading as defined in ( a ) or ( d ) stratification of cancer cases according to ERG ‐fusion status. ( b – d ) Data represent mean + SEM. Number of patients analyzed is indicated ( n ). Statistical significance is shown (* p

    Article Snippet: Consistent with our previous findings that Nox4 is strongly upregulated in activated fibroblasts, significantly higher numbers of Nox4 mRNA‐expressing cells (0.73%, p < 0.001) and an abundance of densely stained clusters (denoting the presence of multiple transcripts/cell) were observed in PCa, particularly within the peritumoral stroma (Supporting Information, Fig. S2).

    Techniques: Expressing, Staining, In Situ Hybridization

    Elevated Nox4 mRNA in the PCa‐associated stroma is associated with epithelial TGFβ expression. Dual Nox4 in situ hybridization and TGFβ immunohistochemistry (pan TGFβ antibody) on radical prostatectomy prostate tissue from PCa patients. Two cancer (CA) and one benign (BE) adjacent areas are shown. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Yellow/brown chromogen staining for TGFβ. Original magnification: top panel 20×, middle and bottom panels 40×. Images are representative of 3 independent experiments using tissue isolated from different patients. Middle right panel: *, benign gland expressing very low levels of TGFβ and little stromal Nox4 with adjacent TGFβ‐positive tumor cells (#) surrounded by abundant Nox4 expression in the tumor‐associated stroma. Black and red boxed regions are enlarged in the middle and lower panels, respectively. Images of parallel‐stained negative controls are depicted in Supporting Information, Figure S6.

    Journal: International Journal of Cancer

    Article Title: Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

    doi: 10.1002/ijc.31316

    Figure Lengend Snippet: Elevated Nox4 mRNA in the PCa‐associated stroma is associated with epithelial TGFβ expression. Dual Nox4 in situ hybridization and TGFβ immunohistochemistry (pan TGFβ antibody) on radical prostatectomy prostate tissue from PCa patients. Two cancer (CA) and one benign (BE) adjacent areas are shown. Single and multiple Nox4 mRNA transcripts appear as single red dots or clusters, respectively. Yellow/brown chromogen staining for TGFβ. Original magnification: top panel 20×, middle and bottom panels 40×. Images are representative of 3 independent experiments using tissue isolated from different patients. Middle right panel: *, benign gland expressing very low levels of TGFβ and little stromal Nox4 with adjacent TGFβ‐positive tumor cells (#) surrounded by abundant Nox4 expression in the tumor‐associated stroma. Black and red boxed regions are enlarged in the middle and lower panels, respectively. Images of parallel‐stained negative controls are depicted in Supporting Information, Figure S6.

    Article Snippet: Consistent with our previous findings that Nox4 is strongly upregulated in activated fibroblasts, significantly higher numbers of Nox4 mRNA‐expressing cells (0.73%, p < 0.001) and an abundance of densely stained clusters (denoting the presence of multiple transcripts/cell) were observed in PCa, particularly within the peritumoral stroma (Supporting Information, Fig. S2).

    Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Isolation

    PCa cell‐derived TGFβ induces fibroblast activation in a paracrine manner dependent on stromal Nox4. ( a ) ELISA quantification of TGFβ1 or TGFβ2 in serum‐free conditioned media (CM) harvested from the indicated prostate epithelial cell line after 72 hr and normalized against non‐CM (serum‐free media incubated without cells). Data represent mean concentration + SEM from duplicate measurements of 3 independent experiments using 3 different CM isolates. qPCR ( b ) or Western blotting ( c ) of prostate fibroblasts pretreated with 1 μM TGFβR inhibitor SB431452 (TGFβRi), 30 μM Nox4 inhibitor GKT137831 (Nox4i) or vehicle equivalent (control, ctrl) in serum‐free DMEM before addition of PC3 PCa cell CM or non‐CM control (serum‐free media incubated without cells). ( b ) Data represent mean fold change + SEM in gene expression relative to the housekeeping gene TBP from three independent experiments using primary fibroblasts isolated from different donors. Statistical significance is shown (* p

    Journal: International Journal of Cancer

    Article Title: Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

    doi: 10.1002/ijc.31316

    Figure Lengend Snippet: PCa cell‐derived TGFβ induces fibroblast activation in a paracrine manner dependent on stromal Nox4. ( a ) ELISA quantification of TGFβ1 or TGFβ2 in serum‐free conditioned media (CM) harvested from the indicated prostate epithelial cell line after 72 hr and normalized against non‐CM (serum‐free media incubated without cells). Data represent mean concentration + SEM from duplicate measurements of 3 independent experiments using 3 different CM isolates. qPCR ( b ) or Western blotting ( c ) of prostate fibroblasts pretreated with 1 μM TGFβR inhibitor SB431452 (TGFβRi), 30 μM Nox4 inhibitor GKT137831 (Nox4i) or vehicle equivalent (control, ctrl) in serum‐free DMEM before addition of PC3 PCa cell CM or non‐CM control (serum‐free media incubated without cells). ( b ) Data represent mean fold change + SEM in gene expression relative to the housekeeping gene TBP from three independent experiments using primary fibroblasts isolated from different donors. Statistical significance is shown (* p

    Article Snippet: Consistent with our previous findings that Nox4 is strongly upregulated in activated fibroblasts, significantly higher numbers of Nox4 mRNA‐expressing cells (0.73%, p < 0.001) and an abundance of densely stained clusters (denoting the presence of multiple transcripts/cell) were observed in PCa, particularly within the peritumoral stroma (Supporting Information, Fig. S2).

    Techniques: Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Isolation

    NOS detection in chromatophore organs of S. officinalis . A–C , NADPH diaphorase reaction. A , embryo chromatophore organs. The NADPH diaphorase staining is visible in the cytoelastic sacculus, which is not fully pigmented. B , isolated juvenile chromatophore organ showing the staining in the fully pigmented sacculus and in the relaxed and contracted muscle fibers. C , isolated adult chromatophore organ with the reaction localized in contracted muscle fibers. D–F , controls of embryo, juvenile, and adult chromatophore organs with omission of β-NADPH showing loss of reaction and solubilization of embryo and juvenile chromatophore pigment ( D and E ). G–I , immunohistochemistry/cytochemistry reaction. G , embryo chromatophore organs. The immunopositivity is localized in the cytoelastic sacculus. H , isolated juvenile chromatophore organ showing the immunoreactivity in the pigment sacculus and in the contracted and partially relaxed muscle fibers. I , Sepia isolated adult chromatophore organ. The immunopositivity is visible in contracted and partially relaxed muscle fibers. Scale bars , 20 μm. mf , muscle fiber; sa , sacculus.

    Journal: The Journal of Biological Chemistry

    Article Title: Nitric Oxide Mediates the Glutamate-dependent Pathway for Neurotransmission in Sepia officinalis

    doi: 10.1074/jbc.M109.083428

    Figure Lengend Snippet: NOS detection in chromatophore organs of S. officinalis . A–C , NADPH diaphorase reaction. A , embryo chromatophore organs. The NADPH diaphorase staining is visible in the cytoelastic sacculus, which is not fully pigmented. B , isolated juvenile chromatophore organ showing the staining in the fully pigmented sacculus and in the relaxed and contracted muscle fibers. C , isolated adult chromatophore organ with the reaction localized in contracted muscle fibers. D–F , controls of embryo, juvenile, and adult chromatophore organs with omission of β-NADPH showing loss of reaction and solubilization of embryo and juvenile chromatophore pigment ( D and E ). G–I , immunohistochemistry/cytochemistry reaction. G , embryo chromatophore organs. The immunopositivity is localized in the cytoelastic sacculus. H , isolated juvenile chromatophore organ showing the immunoreactivity in the pigment sacculus and in the contracted and partially relaxed muscle fibers. I , Sepia isolated adult chromatophore organ. The immunopositivity is visible in contracted and partially relaxed muscle fibers. Scale bars , 20 μm. mf , muscle fiber; sa , sacculus.

    Article Snippet: l -Glutamic acid, diethylamine (DEA), ruthenium red, cADP ribose, 8-bromo-cyclic ADP-ribose (8-Br-cADP ribose), 5-HT, 1,3,7-trimethylxanthine (caffeine), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N -methyl- d -aspartic acid (NMDA), glycine, β-NADPH, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma.

    Techniques: Staining, Isolation, Immunohistochemistry

    Increased HO-1 expression leads to inhibition of PDGF-stimulated NADPH oxidase activity

    Journal:

    Article Title: HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

    doi: 10.1161/ATVBAHA.109.197822

    Figure Lengend Snippet: Increased HO-1 expression leads to inhibition of PDGF-stimulated NADPH oxidase activity

    Article Snippet: Tricarbonyldichlororuthenium (II) dimer (CORM-2), ruthenium (III) chloride hydrate (RuCl3 ), peg-SOD, platelet-derived growth factor BB (PDGF-BB), dihydroethidium (DHE), Triton X-100, dimethyl sulfoxide, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were from Sigma (St. Louis, MO).

    Techniques: Expressing, Inhibition, Activity Assay

    CO inhibits PDGF-stimulated NADPH oxidase activity

    Journal:

    Article Title: HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

    doi: 10.1161/ATVBAHA.109.197822

    Figure Lengend Snippet: CO inhibits PDGF-stimulated NADPH oxidase activity

    Article Snippet: Tricarbonyldichlororuthenium (II) dimer (CORM-2), ruthenium (III) chloride hydrate (RuCl3 ), peg-SOD, platelet-derived growth factor BB (PDGF-BB), dihydroethidium (DHE), Triton X-100, dimethyl sulfoxide, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were from Sigma (St. Louis, MO).

    Techniques: Activity Assay

    Anti-angiogenic therapy targeting NADPH oxidase (ROS)-dependent VEGF signaling

    Journal: Cancer letters

    Article Title: Reactive Oxygen Species and Angiogenesis: NADPH Oxidase as Target for Cancer Therapy

    doi: 10.1016/j.canlet.2008.02.044

    Figure Lengend Snippet: Anti-angiogenic therapy targeting NADPH oxidase (ROS)-dependent VEGF signaling

    Article Snippet: Ushio-Fukai M. Localizing NADPH oxidase-derived ROS.

    Techniques:

    Role of ROS derived from NADPH oxidase in VEGF signaling linked to induction of transcription factors and genes involved in angiogenesis

    Journal: Cancer letters

    Article Title: Reactive Oxygen Species and Angiogenesis: NADPH Oxidase as Target for Cancer Therapy

    doi: 10.1016/j.canlet.2008.02.044

    Figure Lengend Snippet: Role of ROS derived from NADPH oxidase in VEGF signaling linked to induction of transcription factors and genes involved in angiogenesis

    Article Snippet: Ushio-Fukai M. Localizing NADPH oxidase-derived ROS.

    Techniques: Derivative Assay

    GAGs from paw/ankle or knee/elbow were partial inhibitors of enzymatic activity of GPI. Each well contained 4.2 units/ml glucose-6-phosphate dehydrogenase, 1.0 m m NADP, 0.4 μg/ml (6.3 μ m ) recombinant mouse GPI, and inhibitors at various

    Journal:

    Article Title: High Affinity Glycosaminoglycan and Autoantigen Interaction Explains Joint Specificity in a Mouse Model of Rheumatoid Arthritis *

    doi: 10.1074/jbc.M806458200

    Figure Lengend Snippet: GAGs from paw/ankle or knee/elbow were partial inhibitors of enzymatic activity of GPI. Each well contained 4.2 units/ml glucose-6-phosphate dehydrogenase, 1.0 m m NADP, 0.4 μg/ml (6.3 μ m ) recombinant mouse GPI, and inhibitors at various

    Article Snippet: At a final dilution, each well contained 4.2 units/ml glucose-6-phosphate dehydrogenase (Sigma G-6378), 1.0 m m NADP (Sigma N0505), 0.4 μg/ml recombinant mouse GPI, and inhibitors at various concentrations in 50 m m Tris, 88 m m KCl, pH 8.0.

    Techniques: Activity Assay, Recombinant

    Transcription factors that determine B cell identity regulate activity of the pentose phosphate pathway and dependency on PP2A (A) Human embryonic kidney cells (HEK-293) were transduced with a polycistronic doxycycline (Dox)-inducible vector to express the B-lineage transcription factors Pax5, Ikaros, Ebf1 and Tcf3, as confirmed by Western blot after Dox treatment for the times indicated. (B) Relative NADPH/NADP ratios were measured before and after 24 hours of Dox-induction of the polycistronic vector (Pax5, Ikaros, Ebf1 and Tcf3) or EV. (C) Murine myeloid leukemia cells were transduced with the inducible polycistronic vector for myeloid→B cell reprogramming. Phenotypic changes were monitored by flow cytometry 48 hours after induction of Dox and expression of Pax5, Ikaros, Ebf1 and Tcf3 was confirmed by Western blot. (E) In the same cells, protein levels for PP2A Sub A, PP2A Sub C and G6pdx were measured by Western blot. (F) Human small cell lung cancer (SCLC) and normal lung epithelial (BEAS2B) cell lines were used to compare PAX5, PP2A and G6PD expression by Western blot. (G) NADPH/NADP ratios were measured in PAX5 + and PAX5 − SLCL cell lines and upon expression of PAX5 or EV. (H) Murine Pax5 +/− Ebf1 +/− BCR-ABL1 B-ALL cells were treated with the PP2A-inhibitor LB-100 at the concentrations indicated. Dose response curves depicting relative cell viability after 72 hours are depicted. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Journal: Cell

    Article Title: B cell-specific diversion of glucose carbon utilization reveals a unique vulnerability in B cell malignancies

    doi: 10.1016/j.cell.2018.02.048

    Figure Lengend Snippet: Transcription factors that determine B cell identity regulate activity of the pentose phosphate pathway and dependency on PP2A (A) Human embryonic kidney cells (HEK-293) were transduced with a polycistronic doxycycline (Dox)-inducible vector to express the B-lineage transcription factors Pax5, Ikaros, Ebf1 and Tcf3, as confirmed by Western blot after Dox treatment for the times indicated. (B) Relative NADPH/NADP ratios were measured before and after 24 hours of Dox-induction of the polycistronic vector (Pax5, Ikaros, Ebf1 and Tcf3) or EV. (C) Murine myeloid leukemia cells were transduced with the inducible polycistronic vector for myeloid→B cell reprogramming. Phenotypic changes were monitored by flow cytometry 48 hours after induction of Dox and expression of Pax5, Ikaros, Ebf1 and Tcf3 was confirmed by Western blot. (E) In the same cells, protein levels for PP2A Sub A, PP2A Sub C and G6pdx were measured by Western blot. (F) Human small cell lung cancer (SCLC) and normal lung epithelial (BEAS2B) cell lines were used to compare PAX5, PP2A and G6PD expression by Western blot. (G) NADPH/NADP ratios were measured in PAX5 + and PAX5 − SLCL cell lines and upon expression of PAX5 or EV. (H) Murine Pax5 +/− Ebf1 +/− BCR-ABL1 B-ALL cells were treated with the PP2A-inhibitor LB-100 at the concentrations indicated. Dose response curves depicting relative cell viability after 72 hours are depicted. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Article Snippet: 2×107 cells were harvested and subjected to measure NADPH/NADP ratio by NADP/NADPH Assay Kit (ab65349, Abcam).

    Techniques: Activity Assay, Transduction, Plasmid Preparation, Western Blot, Flow Cytometry, Cytometry, Expressing, Standard Deviation

    The fructose-2,6-bisphosphate 2-phosphatase TIGAR rescues cell death upon PP2A-deletion (A) Schematic diagram of TIGAR and PP2A in balancing glucose carbon flux via glycolysis and PPP. (B) Ppp2r1a fl/fl BCR-ABL1 B-ALL cells carrying inducible Cre or EV were subsequently transduced with Tigar-IRES-Orange (Tigar) or Orange-empty vector (EV). Tigar expression was measured by Western blot. (C) Percentages of Orange + B-ALL cells were monitored in a competitive growth assay following 4-OHT treatment. Representative FACS plots are shown at the times indicated (D). (E) Ppp2r1a fl/fl B-ALL cells carrying inducible Cre or EV were transduced with vectors for reconstitution or overexpression of PP2A Sub A (Sub A) or GFP-EV and assayed for cellular NADPH/NADP ratios after 2-days of 4-OHT treatment for inducible activation of Cre or EV for deletion of Ppp2r1a . Likewise, Orange + Ppp2r1a fl/fl B-ALL cells carrying inducible Cre or EV were transduced with Tigar or EV and assayed for cellular NADPH/NADP ratios after 2-days 4-OHT treatment (F). Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Journal: Cell

    Article Title: B cell-specific diversion of glucose carbon utilization reveals a unique vulnerability in B cell malignancies

    doi: 10.1016/j.cell.2018.02.048

    Figure Lengend Snippet: The fructose-2,6-bisphosphate 2-phosphatase TIGAR rescues cell death upon PP2A-deletion (A) Schematic diagram of TIGAR and PP2A in balancing glucose carbon flux via glycolysis and PPP. (B) Ppp2r1a fl/fl BCR-ABL1 B-ALL cells carrying inducible Cre or EV were subsequently transduced with Tigar-IRES-Orange (Tigar) or Orange-empty vector (EV). Tigar expression was measured by Western blot. (C) Percentages of Orange + B-ALL cells were monitored in a competitive growth assay following 4-OHT treatment. Representative FACS plots are shown at the times indicated (D). (E) Ppp2r1a fl/fl B-ALL cells carrying inducible Cre or EV were transduced with vectors for reconstitution or overexpression of PP2A Sub A (Sub A) or GFP-EV and assayed for cellular NADPH/NADP ratios after 2-days of 4-OHT treatment for inducible activation of Cre or EV for deletion of Ppp2r1a . Likewise, Orange + Ppp2r1a fl/fl B-ALL cells carrying inducible Cre or EV were transduced with Tigar or EV and assayed for cellular NADPH/NADP ratios after 2-days 4-OHT treatment (F). Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Article Snippet: 2×107 cells were harvested and subjected to measure NADPH/NADP ratio by NADP/NADPH Assay Kit (ab65349, Abcam).

    Techniques: Transduction, Plasmid Preparation, Expressing, Western Blot, Growth Assay, FACS, Over Expression, Activation Assay, Standard Deviation

    PP2A is essential in B-lymphoid but not myeloid cells to balance glucose carbon utilization via glycolysis and PPP Extracellular acidification rates (ECAR) were measured in (A) B-lymphoid and (B) myeloid Ppp2r1a fl/fl BCR-ABL1 leukemia cells that were transduced with Cre-ER T2 (Cre) or ER T2 -vector (EV). Cells were treated with 4-OHT for 2 days prior to ECAR measurements. (C-D) Ppp2r1a fl/fl BCR-ABL1 ALL cells (B-lymphoid) were cultured with 25 mmol/l 1,2-2 13 C D-glucose for 24 hours, then harvested to extract metabolites for LC-MS based profiling. Ppp2r1a fl/fl BCR-ABL1 ALL cells carrying Tet On - Cebpa were transduced with Cre or EV and were treated for 5-days with Dox for B→myeloid reprogramming. Cre-induced changes of cellular amounts of metabolites that were utilized in glycolysis (M2 13 C isotopomer) or the pentose phosphate pathway (PPP; M1 13 C isotopomer) are shown on a Log 2 -fold scale (Cre relative to EV) in B-lymphoid (C) and B→myeloid (D) cells. Phosphorylation levels of Pfkfb2 at S-483 were measured by Western blot in Ppp2r1a fl/fl B-lymphoid and myeloid leukemia cells after 4-OHT treatment for Cre-mediated deletion of Ppp2r1a . Asterisks denote non-specific bands. (F) Cellular lactate M1/M2 ratio is measured from metabolites profiling data and indicates the relative amount of glucose carbon utilization via PPP and glycolysis pathways, respectively. Relative cellular F1,6BP/G6P, F6P (G) and GSH/GSSG ratios (I) were calculated in B-lymphoid and B→myeloid cells. (H) Relative NADPH/NADP ratios were measured in Ppp2r1a fl/fl B-lymphoid and myeloid cells after 2-days 4-OHT treatment for Cre-mediated deletion of Ppp2r1a . (J) Western blot to compare PP2A and G6PD expression in patient-derived B-ALL (n=4; MXP2,LAX2, PDX2 and BLQ5), mantle cell lymphoma (MCL; n=3; BOS4, BOS5 and BOS6), diffuse large B cell lymphoma (DLBCL; n=2; BOS10 and BOS12), and chronic myeloid leukemia (n=10; Table S4) samples. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Journal: Cell

    Article Title: B cell-specific diversion of glucose carbon utilization reveals a unique vulnerability in B cell malignancies

    doi: 10.1016/j.cell.2018.02.048

    Figure Lengend Snippet: PP2A is essential in B-lymphoid but not myeloid cells to balance glucose carbon utilization via glycolysis and PPP Extracellular acidification rates (ECAR) were measured in (A) B-lymphoid and (B) myeloid Ppp2r1a fl/fl BCR-ABL1 leukemia cells that were transduced with Cre-ER T2 (Cre) or ER T2 -vector (EV). Cells were treated with 4-OHT for 2 days prior to ECAR measurements. (C-D) Ppp2r1a fl/fl BCR-ABL1 ALL cells (B-lymphoid) were cultured with 25 mmol/l 1,2-2 13 C D-glucose for 24 hours, then harvested to extract metabolites for LC-MS based profiling. Ppp2r1a fl/fl BCR-ABL1 ALL cells carrying Tet On - Cebpa were transduced with Cre or EV and were treated for 5-days with Dox for B→myeloid reprogramming. Cre-induced changes of cellular amounts of metabolites that were utilized in glycolysis (M2 13 C isotopomer) or the pentose phosphate pathway (PPP; M1 13 C isotopomer) are shown on a Log 2 -fold scale (Cre relative to EV) in B-lymphoid (C) and B→myeloid (D) cells. Phosphorylation levels of Pfkfb2 at S-483 were measured by Western blot in Ppp2r1a fl/fl B-lymphoid and myeloid leukemia cells after 4-OHT treatment for Cre-mediated deletion of Ppp2r1a . Asterisks denote non-specific bands. (F) Cellular lactate M1/M2 ratio is measured from metabolites profiling data and indicates the relative amount of glucose carbon utilization via PPP and glycolysis pathways, respectively. Relative cellular F1,6BP/G6P, F6P (G) and GSH/GSSG ratios (I) were calculated in B-lymphoid and B→myeloid cells. (H) Relative NADPH/NADP ratios were measured in Ppp2r1a fl/fl B-lymphoid and myeloid cells after 2-days 4-OHT treatment for Cre-mediated deletion of Ppp2r1a . (J) Western blot to compare PP2A and G6PD expression in patient-derived B-ALL (n=4; MXP2,LAX2, PDX2 and BLQ5), mantle cell lymphoma (MCL; n=3; BOS4, BOS5 and BOS6), diffuse large B cell lymphoma (DLBCL; n=2; BOS10 and BOS12), and chronic myeloid leukemia (n=10; Table S4) samples. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.

    Article Snippet: 2×107 cells were harvested and subjected to measure NADPH/NADP ratio by NADP/NADPH Assay Kit (ab65349, Abcam).

    Techniques: Transduction, Plasmid Preparation, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Derivative Assay, Standard Deviation

    Extract ion ( m / z 956 → 817) chromatograms obtained from LC-LTQ MS analysis of microsomal incubations containing LIM, GSH, NAL, and NADPH in the absence microsomes (a), or in presence of HLMs (b) or MLMs (c). (d) Extracted ion ( m / z 956 → 817) chromatogram obtained from LC-LTQ MS analysis of synthetic M1 and M1′. (e) MS/MS spectrum of M1 generated in microsomal incubations (M1′ showed the same MS/MS spectrum). (f) MS/MS spectrum of synthetic M1 (synthetic M1′ showed the same MS/MS spectrum).

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Detection and Structural Characterization of Nucleophiles Trapped Reactive Metabolites of Limonin Using Liquid Chromatography-Mass Spectrometry

    doi: 10.1155/2018/3797389

    Figure Lengend Snippet: Extract ion ( m / z 956 → 817) chromatograms obtained from LC-LTQ MS analysis of microsomal incubations containing LIM, GSH, NAL, and NADPH in the absence microsomes (a), or in presence of HLMs (b) or MLMs (c). (d) Extracted ion ( m / z 956 → 817) chromatogram obtained from LC-LTQ MS analysis of synthetic M1 and M1′. (e) MS/MS spectrum of M1 generated in microsomal incubations (M1′ showed the same MS/MS spectrum). (f) MS/MS spectrum of synthetic M1 (synthetic M1′ showed the same MS/MS spectrum).

    Article Snippet: Mouse liver microsomes (MLMs), human liver microsomes (HLMs), recombinant human P450 enzymes, NADPH-regenerating system, glutathione (GSH), N -acetyl cysteine (NAC), and N -acetyl lysine (NAL) were purchased from BD Biosciences (Bedford, MA, USA).

    Techniques: Mass Spectrometry, Generated

    Extract ion ( m / z 802 → 758) chromatograms obtained from LC-LTQ MS analysis of microsomal incubations containing LIM, GSH, NAL, and NADPH in the absence microsomes (a) or in presence of HLMs (b) or MLMs (c). (d) Extracted ion ( m / z 802 → 758) chromatogram obtained from LC-LTQ MS analysis of synthetic M5 and M5′. (e) MS/MS spectrum of M5 generated in microsomal incubations (M5′ showed the same MS/MS spectrum). (f) MS/MS spectrum of synthetic M1 (synthetic M1′ showed the same MS/MS spectrum).

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Detection and Structural Characterization of Nucleophiles Trapped Reactive Metabolites of Limonin Using Liquid Chromatography-Mass Spectrometry

    doi: 10.1155/2018/3797389

    Figure Lengend Snippet: Extract ion ( m / z 802 → 758) chromatograms obtained from LC-LTQ MS analysis of microsomal incubations containing LIM, GSH, NAL, and NADPH in the absence microsomes (a) or in presence of HLMs (b) or MLMs (c). (d) Extracted ion ( m / z 802 → 758) chromatogram obtained from LC-LTQ MS analysis of synthetic M5 and M5′. (e) MS/MS spectrum of M5 generated in microsomal incubations (M5′ showed the same MS/MS spectrum). (f) MS/MS spectrum of synthetic M1 (synthetic M1′ showed the same MS/MS spectrum).

    Article Snippet: Mouse liver microsomes (MLMs), human liver microsomes (HLMs), recombinant human P450 enzymes, NADPH-regenerating system, glutathione (GSH), N -acetyl cysteine (NAC), and N -acetyl lysine (NAL) were purchased from BD Biosciences (Bedford, MA, USA).

    Techniques: Mass Spectrometry, Generated

    Redox homeostasis in SKOV3/DDP cells is maintained intrinsically by pairing oxidative phosphorylation with pentose phosphate pathway. (A) Cellular NADPH content and (B) NADPH/NADP + ratio, (C) GSH and (D) GSSG contents, (E) total GSH (GSH plus GSSG) level and (F) GSH/GSSG ratio determined using enzymatic assays. (G) The expression level G6PD gene was detected using reverse transcription-quantitative polymerase chain reaction. (H) G6PD protein expression level was determined using western blotting, and the enzymatic activity was analyzed using a G6PD assay kit. The data are representative of three experiments. (I) Cell viability of SKOV3 or SKOV3/DDP cells was determined using a MTT assay in the presence of G6PD inhibitors (20 μ M 6-AN or 250 μ M DHEA) with or without cisplatin for 24 h. * P

    Journal: International Journal of Oncology

    Article Title: ABT737 reverses cisplatin resistance by targeting glucose metabolism of human ovarian cancer cells

    doi: 10.3892/ijo.2018.4476

    Figure Lengend Snippet: Redox homeostasis in SKOV3/DDP cells is maintained intrinsically by pairing oxidative phosphorylation with pentose phosphate pathway. (A) Cellular NADPH content and (B) NADPH/NADP + ratio, (C) GSH and (D) GSSG contents, (E) total GSH (GSH plus GSSG) level and (F) GSH/GSSG ratio determined using enzymatic assays. (G) The expression level G6PD gene was detected using reverse transcription-quantitative polymerase chain reaction. (H) G6PD protein expression level was determined using western blotting, and the enzymatic activity was analyzed using a G6PD assay kit. The data are representative of three experiments. (I) Cell viability of SKOV3 or SKOV3/DDP cells was determined using a MTT assay in the presence of G6PD inhibitors (20 μ M 6-AN or 250 μ M DHEA) with or without cisplatin for 24 h. * P

    Article Snippet: Then, the cells were subjected to analysis with the NADP+ /NADPH Quantification kit (BioVision, Inc., Milpitas, CA, USA) to determine NADPH levels and NADPH/NADP+ ratios.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, G6PD Assay, MTT Assay

    SPINK1 regulates the redox status of NSCLC cells. The inhibition of SPINK1 expression significantly increased oxidized DCF level (ROS production), increased the NADP/NADPH ratio, decreased GSH/GSSG ratio and decreased JC-1 aggregates in (A) A549 and (B) H1299 cells. *P

    Journal: Oncology Letters

    Article Title: SPINK1 is a prognosis predicting factor of non-small cell lung cancer and regulates redox homeostasis

    doi: 10.3892/ol.2019.11005

    Figure Lengend Snippet: SPINK1 regulates the redox status of NSCLC cells. The inhibition of SPINK1 expression significantly increased oxidized DCF level (ROS production), increased the NADP/NADPH ratio, decreased GSH/GSSG ratio and decreased JC-1 aggregates in (A) A549 and (B) H1299 cells. *P

    Article Snippet: The intracellular ATP level, NADPH/NADP+ ratio and NADH/NAD+ ratio was evaluated using the ATP Assay kit (Beyotime Institute of Biotechnology), the NADP/NADPH Quantitation Colorimetric kit (BioVision) and the NAD/NADH Quantitation Colorimetric kit (BioVision), according to the manufacturers' protocols.

    Techniques: Inhibition, Expressing

    Effect of RNAi targeting G6PD, PGD and ME, in M . alpina . The expression level ( a ), enzymatic activity ( b ), NADPH level ( c ) and total fatty acid level ( d ) in M. alpina strains after RNAi targeting ME, G6PD and PGD. M. alpina (up-triangles), wild type M. alpina . MA-ME1-S1 (open circle), M. alpina malic enzyme 1 silenced strain; MA-ME2-S1 (open squar e), M. alpina malic enzyme 2 silenced strain; MA-G6PD-S1 (circles), M. alpina glucose-6-phosphate dehydrogenase co-silenced strain; MA-PGD-S1 (squares), M. alpina phosphogluconate dehydrogenase silenced strain. Strains were cultured and sampled as performed in the transcriptome experiment above. Three independent experiments were performed, and the error bars represent standard deviations. * p

    Journal: Scientific Reports

    Article Title: Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi

    doi: 10.1038/srep11247

    Figure Lengend Snippet: Effect of RNAi targeting G6PD, PGD and ME, in M . alpina . The expression level ( a ), enzymatic activity ( b ), NADPH level ( c ) and total fatty acid level ( d ) in M. alpina strains after RNAi targeting ME, G6PD and PGD. M. alpina (up-triangles), wild type M. alpina . MA-ME1-S1 (open circle), M. alpina malic enzyme 1 silenced strain; MA-ME2-S1 (open squar e), M. alpina malic enzyme 2 silenced strain; MA-G6PD-S1 (circles), M. alpina glucose-6-phosphate dehydrogenase co-silenced strain; MA-PGD-S1 (squares), M. alpina phosphogluconate dehydrogenase silenced strain. Strains were cultured and sampled as performed in the transcriptome experiment above. Three independent experiments were performed, and the error bars represent standard deviations. * p

    Article Snippet: The NADP and NADPH levels were determined using the NADP/NADPH Quantification Colorimetric Kit (BioVision, California, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Activity Assay, Cell Culture

    Nutlin inhibits or promotes PPP. ( A ) Cells were treated with vehicle or Nutlin (10 μM) and/or MK2206 (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated. ( B ) Cells were transfected with control siRNA, TIGAR siRNA, or G6PD siRNA and then treated with vehicle or Nutlin (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated (left panel). Lysates were also immunoblotted for the indicated proteins (right panel). ( C ) cells were treated with Nutlin for a 12-h period and then fluxed with D-[1,2- 13 C] glucose for 15 min. Metabolites were analyzed by LC–MS. Ribose-5-P- 13 C1 levels in vehicle-treated and Nutlin-treated cells were presented with SD indicated. There is no significant difference between vehicle and Nutlin in U2OS cells ( P = 0.22). There is significant difference between vehicle and Nutlin in MHM cells ( P = 0.008). ( D ) Metabolism of 1,2- 13 C 2 -glucose through PPP to generate NADPH and into ribose-5-P- 13 C1 is schematically presented.

    Journal: Journal of Molecular Cell Biology

    Article Title: p53 promotes AKT and SP1-dependent metabolism through the pentose phosphate pathway that inhibits apoptosis in response to Nutlin-3a

    doi: 10.1093/jmcb/mjx051

    Figure Lengend Snippet: Nutlin inhibits or promotes PPP. ( A ) Cells were treated with vehicle or Nutlin (10 μM) and/or MK2206 (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated. ( B ) Cells were transfected with control siRNA, TIGAR siRNA, or G6PD siRNA and then treated with vehicle or Nutlin (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated (left panel). Lysates were also immunoblotted for the indicated proteins (right panel). ( C ) cells were treated with Nutlin for a 12-h period and then fluxed with D-[1,2- 13 C] glucose for 15 min. Metabolites were analyzed by LC–MS. Ribose-5-P- 13 C1 levels in vehicle-treated and Nutlin-treated cells were presented with SD indicated. There is no significant difference between vehicle and Nutlin in U2OS cells ( P = 0.22). There is significant difference between vehicle and Nutlin in MHM cells ( P = 0.008). ( D ) Metabolism of 1,2- 13 C 2 -glucose through PPP to generate NADPH and into ribose-5-P- 13 C1 is schematically presented.

    Article Snippet: Cells were lysed and cellular NADPH was quantified according to the manufacturer’s guidelines using NADP/NADPH Quantification Kit (Sigma-Altrich).

    Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy

    MJ25 is an inhibitor of thioredoxin reductase 1 (TrxR1) a. The capability of MJ25 and auranofin to inhibit recombinant, rat-derived TrxR1 in vitro was measured by an NADPH dependent 5,5′-dithiobis-[2-nitrobenzoic acid] (DTNB) assay. b. ARN8 cells were treated with MJ25, auranofin or DMSO, respectively, for the indicated periods of time. TrxR1 inhibition was subsequently assessed in cell lysates by an NADPH and Trx dependent insulin reduction endpoint assay, measuring thiol formation using DTNB. Ratios between MJ25 and DMSO as well as auranofin and DMSO were determined for each point in time. (c and d) ARN8 cells were treated with c. MJ25 or d. auranofin, while in each half of the samples growth media were supplemented with sodium selenite [75 nM] three days prior to seeding as well as during seeding and treatment for 72 hours. Cell viability and clonogenic capacity were determined. e. ROS levels were determined in ARN8 cells 3 hours after the indicated treatment by measuring fluorescence of 2′,7′-dichlorofluorescein (DCF). f. Induction of anti-oxidative proteins by MJ25 and auranofin was investigated in ARN8 cells at the indicated points in time by Western blotting. DMSO served as vehicle control (0 μM). g. ARN8 cells were pre-treated with L-buthionine sulfoximine (BSO) or vehicle (H 2 O) for 72 hours, upon which cells were re-plated in BSO- and vehicle-free growth medium. Cell viability was assessed by SRB assay after 72 hours in the presence of vehicle (DMSO), MJ25 (left panel) or auranofin (middle panel), respectively. Intracellular glutathione (GSH) levels were determined 72 hours after BSO / vehicle treatment (right panel). h. Inhibition of yeast-derived glutathione reductase by MJ25 and auranofin was determined in vitro by measurement of glutathione disulfide (GSSG) reduction.

    Journal: Oncotarget

    Article Title: Redox effects and cytotoxic profiles of MJ25 and auranofin towards malignant melanoma cells

    doi:

    Figure Lengend Snippet: MJ25 is an inhibitor of thioredoxin reductase 1 (TrxR1) a. The capability of MJ25 and auranofin to inhibit recombinant, rat-derived TrxR1 in vitro was measured by an NADPH dependent 5,5′-dithiobis-[2-nitrobenzoic acid] (DTNB) assay. b. ARN8 cells were treated with MJ25, auranofin or DMSO, respectively, for the indicated periods of time. TrxR1 inhibition was subsequently assessed in cell lysates by an NADPH and Trx dependent insulin reduction endpoint assay, measuring thiol formation using DTNB. Ratios between MJ25 and DMSO as well as auranofin and DMSO were determined for each point in time. (c and d) ARN8 cells were treated with c. MJ25 or d. auranofin, while in each half of the samples growth media were supplemented with sodium selenite [75 nM] three days prior to seeding as well as during seeding and treatment for 72 hours. Cell viability and clonogenic capacity were determined. e. ROS levels were determined in ARN8 cells 3 hours after the indicated treatment by measuring fluorescence of 2′,7′-dichlorofluorescein (DCF). f. Induction of anti-oxidative proteins by MJ25 and auranofin was investigated in ARN8 cells at the indicated points in time by Western blotting. DMSO served as vehicle control (0 μM). g. ARN8 cells were pre-treated with L-buthionine sulfoximine (BSO) or vehicle (H 2 O) for 72 hours, upon which cells were re-plated in BSO- and vehicle-free growth medium. Cell viability was assessed by SRB assay after 72 hours in the presence of vehicle (DMSO), MJ25 (left panel) or auranofin (middle panel), respectively. Intracellular glutathione (GSH) levels were determined 72 hours after BSO / vehicle treatment (right panel). h. Inhibition of yeast-derived glutathione reductase by MJ25 and auranofin was determined in vitro by measurement of glutathione disulfide (GSSG) reduction.

    Article Snippet: Reduced glutathione (GSH; no. A2084.0005), oxidized glutathione (GSSG; no. G4376), and NADPH (no. A1395.0500) were purchased from AppliChem (Kongens Lyngby, Denmark).

    Techniques: Recombinant, Derivative Assay, In Vitro, DTNB Assay, Inhibition, End Point Assay, Fluorescence, Western Blot, Sulforhodamine B Assay

    Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced NADP and NAD ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4

    doi: 10.1038/cddis.2017.48

    Figure Lengend Snippet: Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced NADP and NAD ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P

    Article Snippet: Biochemical assays Quantification of NAD and NADP was performed according to the manufacturer's instructions (Abcam, ab65348) and (Abcam, ab65349), respectively.

    Techniques: Cell Culture, Transfection, Western Blot, Derivative Assay

    Lsd1 regulates anaplerosis and maintains redox balance. ( a – m ) Comparison of TSCs cultured for 24 h in the presence of solvent (Ctrl), Lsd1 inhibitor 1 (Lsd1 i-1 ) ( a – m ), and Lsd1 inhibitor 2 (Lsd1 i-2 ) ( b – m ). ( a ) Metabolomics profile depicted by log 2 fold change versus −log 10 P -value. Schema and bar graph depicting increased (red) and decreased (blue) metabolites of glycolysis, TCA cycle and glutamine anaplerosis. ( b – e ) Quantification of glycolysis rate by extracellular acidification rate (ECAR) ( b ), relative glutamine uptake ( c ), Glud1 activity ( d ) and ammonia levels ( e ). ( f ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( g ) Relative mitochondrial membrane potential determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( h – m ) Measurement of ATP concentration ( h ), relative H 2 O 2 concentration determined with cytoplasmic and mitochondrial ratiometric reporters ( i ), relative quantification of ROS using fluorescent dye ( j ), relative concentration of oxidized glutathione (GSSG) determined by cytoplasmic and mitochondrial ratiometric reporters ( k ), relative ratio of oxidized to reduced NADP and NAD ( l ) and relative glutathione levels ( m ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4

    doi: 10.1038/cddis.2017.48

    Figure Lengend Snippet: Lsd1 regulates anaplerosis and maintains redox balance. ( a – m ) Comparison of TSCs cultured for 24 h in the presence of solvent (Ctrl), Lsd1 inhibitor 1 (Lsd1 i-1 ) ( a – m ), and Lsd1 inhibitor 2 (Lsd1 i-2 ) ( b – m ). ( a ) Metabolomics profile depicted by log 2 fold change versus −log 10 P -value. Schema and bar graph depicting increased (red) and decreased (blue) metabolites of glycolysis, TCA cycle and glutamine anaplerosis. ( b – e ) Quantification of glycolysis rate by extracellular acidification rate (ECAR) ( b ), relative glutamine uptake ( c ), Glud1 activity ( d ) and ammonia levels ( e ). ( f ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( g ) Relative mitochondrial membrane potential determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( h – m ) Measurement of ATP concentration ( h ), relative H 2 O 2 concentration determined with cytoplasmic and mitochondrial ratiometric reporters ( i ), relative quantification of ROS using fluorescent dye ( j ), relative concentration of oxidized glutathione (GSSG) determined by cytoplasmic and mitochondrial ratiometric reporters ( k ), relative ratio of oxidized to reduced NADP and NAD ( l ) and relative glutathione levels ( m ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P

    Article Snippet: Biochemical assays Quantification of NAD and NADP was performed according to the manufacturer's instructions (Abcam, ab65348) and (Abcam, ab65349), respectively.

    Techniques: Cell Culture, Activity Assay, Derivative Assay, Concentration Assay

    The central carbon metabolism (CCM). The CCM combines enzymatic reactions that convert carbon sources into biomass precursors. This figure shows the five main pathways forming the CCM: glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle (TCA), lipogenesis, and beta-oxidation. Two opposite metabolic demands are at the core of the CCM: anabolic reactions, which consist in biomass synthesis, and catabolic reactions, leading to the breakdown of macromolecules for energetic use. These two aspects of cell metabolism are managed by biochemical oscillators, including redox couples, such as nicotinamide adenine dinucleotide (NAD + /NADH) and nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD + /NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP + /NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP + /NADPH ratio triggers lipogenesis. The ATP/(ADP + Pi) ratio senses the metabolic state of the cell and may lead to metabolic switches in the CCM.

    Journal: Metabolites

    Article Title: The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect

    doi: 10.3390/metabo6040033

    Figure Lengend Snippet: The central carbon metabolism (CCM). The CCM combines enzymatic reactions that convert carbon sources into biomass precursors. This figure shows the five main pathways forming the CCM: glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle (TCA), lipogenesis, and beta-oxidation. Two opposite metabolic demands are at the core of the CCM: anabolic reactions, which consist in biomass synthesis, and catabolic reactions, leading to the breakdown of macromolecules for energetic use. These two aspects of cell metabolism are managed by biochemical oscillators, including redox couples, such as nicotinamide adenine dinucleotide (NAD + /NADH) and nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD + /NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP + /NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP + /NADPH ratio triggers lipogenesis. The ATP/(ADP + Pi) ratio senses the metabolic state of the cell and may lead to metabolic switches in the CCM.

    Article Snippet: All procedures followed the kit instructions: ENLITEN® ATP Assay System (Promega FF2000; Charbonnières, France) for ATP quantification, NADP+ /NADPH and NAD+ /NADH assays were run using the NADP/NADPH-Glo Bioluminescent Assay kit (Promega G9082) and the NAD/NADH-Glo Bioluminescent Assay kit (Promega G9072), respectively.

    Techniques:

    Activity of thioredoxin (Trx) system enzymes as measured by the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and insulin reduction assay. ( a ) Recovery of selenoenzyme thioredoxin reductase (TrxR, 20 nM) activity inhibited by methylmercury (MeHg, 200 nM) by the chelating agents [15]aneN 4 S, BAL and DMSA (40 µM) as measured by the DTNB reduction assay. ( b ) Recovery of Trx system activity by chelating agents ([15]aneN 4 S, BAL and DMSA; 40 µM) following incubation of TrxR (20 nM), Trx (3 µM) and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) with MeHg (200 nM) as measured by the insulin reduction assay. All data points represent the mean ± SE of at least three independent experiments. * Different from control, p

    Journal: International Journal of Environmental Research and Public Health

    Article Title: In Vitro Assessment of the Efficacy of a Macrocyclic Chelator in Reversing Methylmercury Toxicity

    doi: 10.3390/ijerph16234817

    Figure Lengend Snippet: Activity of thioredoxin (Trx) system enzymes as measured by the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and insulin reduction assay. ( a ) Recovery of selenoenzyme thioredoxin reductase (TrxR, 20 nM) activity inhibited by methylmercury (MeHg, 200 nM) by the chelating agents [15]aneN 4 S, BAL and DMSA (40 µM) as measured by the DTNB reduction assay. ( b ) Recovery of Trx system activity by chelating agents ([15]aneN 4 S, BAL and DMSA; 40 µM) following incubation of TrxR (20 nM), Trx (3 µM) and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) with MeHg (200 nM) as measured by the insulin reduction assay. All data points represent the mean ± SE of at least three independent experiments. * Different from control, p

    Article Snippet: Reagents Methylmercury chloride (CH3 HgCl), dithiothreitol (DTT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), insulin (human recombinant), BAL and DMSA were purchased from Sigma-Aldrich.

    Techniques: Activity Assay, Insulin Reduction Assay, Incubation

    Physiological aspects of antimicrobial activity by HD-6. ( a ) Oxidized HD-6 (top row) or truncated ΔoxHD-6 (bottom row) were incubated with rat thioredoxin reductase, NADPH, and with or without 3 μ M thioredoxin (TRX). Enzymatic conversion from oxidized HD-6 into the reduced form was investigated by RP-HPLC analysis. Representative experiments of three independent experiments are shown. ( b ) Schematic model describing a potential mechanism of HD-6 activity. HD-6 might be secreted as an oxidized peptide by Paneth cells into the crypt, trap bacteria by formation of nanonets that further diffuse toward the lumen. The reducing milieu might reduce HD-6 that eventually kills bacteria. For more details please see text. ( c ) Bif. adolescentis Ni3,29c bacteria were incubated in the absence (untreated) or presence of oxidized HD-6 for 30 min without anaerobic environment, followed by 90 min of incubation in an anaerobic environment in the presence or absence of DTT. Aliquots were plated on agar plates and colony-forming units were calculated the next day. Experiments were carried out at least three times; means with s.e.m. are shown. DTT, dithiothreitol; HD-6, α-defensin 6; oxHD-6, oxidized HD-6; redHD-6, reduced HD-6; RP-HPLC, reversed-phase high-performance liquid chromatography; UV, ultraviolet.

    Journal: Mucosal Immunology

    Article Title: Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide

    doi: 10.1038/mi.2014.100

    Figure Lengend Snippet: Physiological aspects of antimicrobial activity by HD-6. ( a ) Oxidized HD-6 (top row) or truncated ΔoxHD-6 (bottom row) were incubated with rat thioredoxin reductase, NADPH, and with or without 3 μ M thioredoxin (TRX). Enzymatic conversion from oxidized HD-6 into the reduced form was investigated by RP-HPLC analysis. Representative experiments of three independent experiments are shown. ( b ) Schematic model describing a potential mechanism of HD-6 activity. HD-6 might be secreted as an oxidized peptide by Paneth cells into the crypt, trap bacteria by formation of nanonets that further diffuse toward the lumen. The reducing milieu might reduce HD-6 that eventually kills bacteria. For more details please see text. ( c ) Bif. adolescentis Ni3,29c bacteria were incubated in the absence (untreated) or presence of oxidized HD-6 for 30 min without anaerobic environment, followed by 90 min of incubation in an anaerobic environment in the presence or absence of DTT. Aliquots were plated on agar plates and colony-forming units were calculated the next day. Experiments were carried out at least three times; means with s.e.m. are shown. DTT, dithiothreitol; HD-6, α-defensin 6; oxHD-6, oxidized HD-6; redHD-6, reduced HD-6; RP-HPLC, reversed-phase high-performance liquid chromatography; UV, ultraviolet.

    Article Snippet: Briefly, 7.5 μM synthetic oxHD-6 or ΔoxHD-6 was incubated with 0.8 mM NADPH (Biomol, Hamburg, Germany), rat TRX reductase (200 nM, IMCO, Stockholm, Sweden), and 3.0 μM human TRX (Sigma Aldrich) or 3.0 μM bovine protein disulfide isomerase (Sigma Aldrich) in 0.1 M potassium phosphate buffer containing 2 mM EDTA at pH 7.0 for 30 min at 37 °C.

    Techniques: Activity Assay, Incubation, High Performance Liquid Chromatography

    Effect of DBTG on expression of NOX4 in BLM-induced rats PF. (a) Representative western-blots of NOX4 and β -actin at 7 days ( n = 3); (b) representative western-blots of NOX4 and β -actin at 14 days ( n = 3); (c) representative western-blots of NOX4 and β -actin at 28 days ( n = 3). The expression of NOX4 was inhibited in DBTG treatment groups contrast with model group. Data were expressed as mean ± SEM, ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Total Glucosides of Danggui Buxue Tang Attenuate BLM-Induced Pulmonary Fibrosis via Regulating Oxidative Stress by Inhibiting NOX4

    doi: 10.1155/2015/645814

    Figure Lengend Snippet: Effect of DBTG on expression of NOX4 in BLM-induced rats PF. (a) Representative western-blots of NOX4 and β -actin at 7 days ( n = 3); (b) representative western-blots of NOX4 and β -actin at 14 days ( n = 3); (c) representative western-blots of NOX4 and β -actin at 28 days ( n = 3). The expression of NOX4 was inhibited in DBTG treatment groups contrast with model group. Data were expressed as mean ± SEM, ∗ p

    Article Snippet: Then blotted membranes were incubated at 4°C overnight with anti-α -SMA (1 : 100, ab5694, Abcam, Cambridge, UK), anti-NOX4 (1 : 5000, ab133303, Abcam, Cambridge, UK), and anti-β -actin (1 : 5000, ab32572, Abcam, Cambridge, UK) antibodies.

    Techniques: Expressing, Western Blot

    DBTG attenuates BLM-induced rats PF via regulating oxidative stress by inhibiting NOX4. In this study, oxidative stress happened in BLM-induced PF. And then, a lot of ROS was synthesized by NOX4 to promote the expression of TGF- β 1 and its downstream protein α -SMA that increased the degree of PF. DBTG could inhibit NOX4 to reduce ROS and control the level of oxidative stress. As the level of oxidative stress was regulated, the expression of TGF- β 1 and expression of α -SMA were both decreased and, at last, the development of PF could be inhibited.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Total Glucosides of Danggui Buxue Tang Attenuate BLM-Induced Pulmonary Fibrosis via Regulating Oxidative Stress by Inhibiting NOX4

    doi: 10.1155/2015/645814

    Figure Lengend Snippet: DBTG attenuates BLM-induced rats PF via regulating oxidative stress by inhibiting NOX4. In this study, oxidative stress happened in BLM-induced PF. And then, a lot of ROS was synthesized by NOX4 to promote the expression of TGF- β 1 and its downstream protein α -SMA that increased the degree of PF. DBTG could inhibit NOX4 to reduce ROS and control the level of oxidative stress. As the level of oxidative stress was regulated, the expression of TGF- β 1 and expression of α -SMA were both decreased and, at last, the development of PF could be inhibited.

    Article Snippet: Then blotted membranes were incubated at 4°C overnight with anti-α -SMA (1 : 100, ab5694, Abcam, Cambridge, UK), anti-NOX4 (1 : 5000, ab133303, Abcam, Cambridge, UK), and anti-β -actin (1 : 5000, ab32572, Abcam, Cambridge, UK) antibodies.

    Techniques: Synthesized, Expressing

    Metabolism of nicotine by recombinantly expressed aphid (CYP6CY3) and human (CYP2A6) P450s. ( A ) The recovery of nicotine after 10 μM nicotine was incubated with 50 pmol P450+reductase in the presence or absence of NADPH for 60 min. Mean data ±

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Gene amplification and microsatellite polymorphism underlie a recent insect host shift

    doi: 10.1073/pnas.1314122110

    Figure Lengend Snippet: Metabolism of nicotine by recombinantly expressed aphid (CYP6CY3) and human (CYP2A6) P450s. ( A ) The recovery of nicotine after 10 μM nicotine was incubated with 50 pmol P450+reductase in the presence or absence of NADPH for 60 min. Mean data ±

    Article Snippet: Metabolism of nicotine and neonicotinoids (imidacloprid and clothianidin) was assayed by incubating recombinant CYP6CY3/CPR (2 pmol P450 per assay) or ctrl-virus/CPR microsomes in 0.1 M potassium phosphate buffer with an NADPH-regenerating system (Promega; 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2 , 0.4 U/mL−1 glucose-6-phosphate dehydrogenase) and substrate (12.5 µM; 0.8–100 µM for enzyme kinetics) at 27 °C for 1 h. The total assay volume was 200 µL using three replicates for each data point.

    Techniques: Incubation

    Overall structure model ( A ) of ecQueF with preQ 0 and NADPH and close-up view of Trp 57 ( B ) and Trp 198 ( C ). Tryptophan is shown in magenta . Other amino acids in the active site and NADPH are shown in blue and yellow , respectively. Cys 190 , preQ 0 , and amino acids having hydrophilic interaction with Trp 198 .

    Journal: The Journal of Biological Chemistry

    Article Title: Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli *

    doi: 10.1074/jbc.M116.747014

    Figure Lengend Snippet: Overall structure model ( A ) of ecQueF with preQ 0 and NADPH and close-up view of Trp 57 ( B ) and Trp 198 ( C ). Tryptophan is shown in magenta . Other amino acids in the active site and NADPH are shown in blue and yellow , respectively. Cys 190 , preQ 0 , and amino acids having hydrophilic interaction with Trp 198 .

    Article Snippet: NADPH (purity > 98%) and NADP+ (purity > 97%) were from Carl Roth (Karlsruhe, Germany).

    Techniques:

    (A and B) MS analysis of the alcohol product from an incubation of 4-pyridine carboxaldehyde, enzyme, and either NADPH (A) or (4 R )-[4- 2 H]NADPD (B). The corresponding GC traces can be seen in Fig. S2 in the supplemental material. (C) NMR spectra showing the purified nucleotide reaction product obtained from incubations of Cbei_3974 with (4 R )-[4- 2 H]NADPD and 4-pyridine carboxaldehyde (red) against a standard of NADP + (blue).

    Journal: Applied and Environmental Microbiology

    Article Title: Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii

    doi: 10.1128/AEM.00978-19

    Figure Lengend Snippet: (A and B) MS analysis of the alcohol product from an incubation of 4-pyridine carboxaldehyde, enzyme, and either NADPH (A) or (4 R )-[4- 2 H]NADPD (B). The corresponding GC traces can be seen in Fig. S2 in the supplemental material. (C) NMR spectra showing the purified nucleotide reaction product obtained from incubations of Cbei_3974 with (4 R )-[4- 2 H]NADPD and 4-pyridine carboxaldehyde (red) against a standard of NADP + (blue).

    Article Snippet: NADPH was obtained from Fisher or Apollo Scientific. (4 R )-[4-2 H]NADPD was prepared from NADP+ (Melford) and isopropanol-d8 (Acros) according to the published protocol ( ).

    Techniques: Mass Spectrometry, Incubation, Nuclear Magnetic Resonance, Purification

    Involvement of ROS in leptin-mediated GATA-4 nuclear translocation. Effect of the NADPH oxidase inhibitor apocynin (1 μmol/L) on leptin-induced GATA-4 nuclear translocation in RASMC. GATA-4 antibody was visualized by CruzFluor 488-conjugated secondary antibody (second panel; green) while Acti-stain 555 phalloidin was used to stain F-actin (third panel; red). DAPI stained the nuclei blue (first panel; blue). The overlay of DAPI, GATA-4, and F-actin (merged) is shown in the right panel. ROS depletion inhibited GATA-4 nuclear translocation.

    Journal: Frontiers in Pharmacology

    Article Title: Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation

    doi: 10.3389/fphar.2015.00240

    Figure Lengend Snippet: Involvement of ROS in leptin-mediated GATA-4 nuclear translocation. Effect of the NADPH oxidase inhibitor apocynin (1 μmol/L) on leptin-induced GATA-4 nuclear translocation in RASMC. GATA-4 antibody was visualized by CruzFluor 488-conjugated secondary antibody (second panel; green) while Acti-stain 555 phalloidin was used to stain F-actin (third panel; red). DAPI stained the nuclei blue (first panel; blue). The overlay of DAPI, GATA-4, and F-actin (merged) is shown in the right panel. ROS depletion inhibited GATA-4 nuclear translocation.

    Article Snippet: Inhibitors such as the selective ROCK inhibitor Y-27632 (10 μmol/L, Sigma Aldrich, MO, USA), the actin depolymerization agent cytochalasin D (1 μmol/L; Calbiochem, CA, USA), the NADPH oxidase inhibitor apocynin (1 μmol/L, 4-Hydroxy-3-methoxyacetophenone, Sigma Aldrich, MO, USA), and the anti-leptin antibody Ob (Y-20) (1 μmol/L; Santa Cruz Biotechnology, CA, USA) were added to the media 60 min before mechanically stretching the RPV or adding leptin.

    Techniques: Translocation Assay, Staining