nadp  (Abcam)

 
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  • 99
    Name:
    NAD NADH Assay Kit Colorimetric
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    Catalog Number:
    ab65348
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    Structured Review

    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

    https://www.bioz.com/result/nadp/product/Abcam
    Average 99 stars, based on 7 article reviews
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    nadp - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4"

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

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.48

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "Metyrapone Alleviates Deleterious Effects of Maternal Food Restriction on Lung Development and Growth of Rat Offspring"

    Article Title: Metyrapone Alleviates Deleterious Effects of Maternal Food Restriction on Lung Development and Growth of Rat Offspring

    Journal: Reproductive Sciences

    doi: 10.1177/1933719114537712

    The effect of corticosterone (CORT) and metyrapone (MTP) treatment on NADPH/NADPt levels in e19 fetal rat lung lipofibroblasts (LIFs). In e19 LIFs, CORT treatment increased the levels of (A) NADPH and (B) NADPH/NADPt. Metyrapone blocked this effect of
    Figure Legend Snippet: The effect of corticosterone (CORT) and metyrapone (MTP) treatment on NADPH/NADPt levels in e19 fetal rat lung lipofibroblasts (LIFs). In e19 LIFs, CORT treatment increased the levels of (A) NADPH and (B) NADPH/NADPt. Metyrapone blocked this effect of

    Techniques Used:

    3) Product Images from "CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION"

    Article Title: CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION

    Journal: Nature communications

    doi: 10.1038/ncomms5753

    Effect of lung endotoxemia on metabolite-sensing pathways known to regulate clock gene expression patterns. Lung homogenates used for these measurements were derived from Microarray Experiment #2. (a,b) Mean levels of NAD + (a) , and NADP + nucleotides (b) in basal and endotoxemic lungs. Each point represents data obtained from pooled lung homogenates (n=3). Blue lines represent lungs from PBS-treated animals and red lines represent lungs from endotoxemic animals. Significance values for each curve are depicted (ANOVA periodogram assuming τ=12 hours). ( c ) Western blot analysis of phospho-rS6 (Ser 235/236) and phospho-AMPKα1/2 (Thr 172) abundance over time in normal and endotoxemic lungs. Each lane represents 25 µg total protein pooled from n=3 lungs. Similar results were obtained in 2 independent time series experiments. ( c,d ) Densitometric measurement of phsopho-rS6/total rS6 ratio ( d ) and phospho-AMPKα/ total AMPKα ratio ( e ). Blue lines represent data from normal lungs and red lines represent data from endotoxemic lungs. A best fit single harmonic cosine curve is depicted where appropriate. For tabular depiction of COSOPT rhythm analyses of NAD + levels, NADP + levels, phsopho-rS6/total rS6 ratio and phospho-AMPKα/ total AMPKα ratio please see Supplementary Data 11 . Full images of the western blots are depicted in Supplementary Fig. 12 . Note that for these experiments data were collected under constant light (LL 12:12) conditions.
    Figure Legend Snippet: Effect of lung endotoxemia on metabolite-sensing pathways known to regulate clock gene expression patterns. Lung homogenates used for these measurements were derived from Microarray Experiment #2. (a,b) Mean levels of NAD + (a) , and NADP + nucleotides (b) in basal and endotoxemic lungs. Each point represents data obtained from pooled lung homogenates (n=3). Blue lines represent lungs from PBS-treated animals and red lines represent lungs from endotoxemic animals. Significance values for each curve are depicted (ANOVA periodogram assuming τ=12 hours). ( c ) Western blot analysis of phospho-rS6 (Ser 235/236) and phospho-AMPKα1/2 (Thr 172) abundance over time in normal and endotoxemic lungs. Each lane represents 25 µg total protein pooled from n=3 lungs. Similar results were obtained in 2 independent time series experiments. ( c,d ) Densitometric measurement of phsopho-rS6/total rS6 ratio ( d ) and phospho-AMPKα/ total AMPKα ratio ( e ). Blue lines represent data from normal lungs and red lines represent data from endotoxemic lungs. A best fit single harmonic cosine curve is depicted where appropriate. For tabular depiction of COSOPT rhythm analyses of NAD + levels, NADP + levels, phsopho-rS6/total rS6 ratio and phospho-AMPKα/ total AMPKα ratio please see Supplementary Data 11 . Full images of the western blots are depicted in Supplementary Fig. 12 . Note that for these experiments data were collected under constant light (LL 12:12) conditions.

    Techniques Used: Expressing, Derivative Assay, Microarray, Western Blot

    4) Product Images from "PCK1 Downregulation Promotes TXNRD1 Expression and Hepatoma Cell Growth via the Nrf2/Keap1 Pathway"

    Article Title: PCK1 Downregulation Promotes TXNRD1 Expression and Hepatoma Cell Growth via the Nrf2/Keap1 Pathway

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00611

    PCK1 suppresses oxidative stress in liver cancer cells. Relative ratios of NADPH to total NADP in PCK1-overexpressing cells (A) , and PCK1-KO cells (B) were measured by using an NADP/NADPH quantification kit (Abcam). Fluorescent labeling of ROS (red) in the cytoplasm, nuclei were stained with DAPI (blue). Scale bar = 10 μm. ROS levels in PCK1-overexpressing cells (C) , and PCK1-KO cells (D) were detected, and the relative fluorescence intensities were determined after normalization to mock-infected or parental cells. Data are represented as the mean ± SD ( n = 3, technical replicates; ** P
    Figure Legend Snippet: PCK1 suppresses oxidative stress in liver cancer cells. Relative ratios of NADPH to total NADP in PCK1-overexpressing cells (A) , and PCK1-KO cells (B) were measured by using an NADP/NADPH quantification kit (Abcam). Fluorescent labeling of ROS (red) in the cytoplasm, nuclei were stained with DAPI (blue). Scale bar = 10 μm. ROS levels in PCK1-overexpressing cells (C) , and PCK1-KO cells (D) were detected, and the relative fluorescence intensities were determined after normalization to mock-infected or parental cells. Data are represented as the mean ± SD ( n = 3, technical replicates; ** P

    Techniques Used: Labeling, Staining, Fluorescence, Infection

    5) Product Images from "IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity"

    Article Title: IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

    Cisplatin exposure of IDH1 MUT HCT116 cells decrease NADPH levels and increase ROS levels, and AGI-5198 attenuates these effects. A ) Cells were incubated in the presence or absence of 1 µM AGI-5198, treated with cisplatin (5 µM), and harvested, prepared, and colorimetrically analyzed for NADP + :NADPH ratios after 72 h. B ) As in A , but cells were treated with cisplatin (25 µM) and analyzed with a fluorometric assay for ROS levels at different time points. * P
    Figure Legend Snippet: Cisplatin exposure of IDH1 MUT HCT116 cells decrease NADPH levels and increase ROS levels, and AGI-5198 attenuates these effects. A ) Cells were incubated in the presence or absence of 1 µM AGI-5198, treated with cisplatin (5 µM), and harvested, prepared, and colorimetrically analyzed for NADP + :NADPH ratios after 72 h. B ) As in A , but cells were treated with cisplatin (25 µM) and analyzed with a fluorometric assay for ROS levels at different time points. * P

    Techniques Used: Incubation

    6) Product Images from "ROS is the major player in regulating altered autophagy and lifespan in sin-3 mutants of C. elegans"

    Article Title: ROS is the major player in regulating altered autophagy and lifespan in sin-3 mutants of C. elegans

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1474312

    Altered redox NADP and glutathione status in sin-3;him-5 worms. (A) Total NADP content in various C. elegans strains in pmole/ml of lysate. (B) Ratio of NADP:NADPH in various strains (C) Total glutathione content in various C. elegans strains in nmole/mg protein. (D) Ratio of GSH:GSSG in different strains. Where N2, wild-type; sin-3;him-5, sin-3(tm1279);him-5(e1490) mutant; sin-3;him-5 Vit. C, sin-3(tm1279);him-5(e1490); him-5(e1490) Vit. C, him-5(e1490) worms maintained on NGM plate supplemented with 10 mM vitamin C. Other mutants are duly indicated on the graph (ns, non-significant; ** P
    Figure Legend Snippet: Altered redox NADP and glutathione status in sin-3;him-5 worms. (A) Total NADP content in various C. elegans strains in pmole/ml of lysate. (B) Ratio of NADP:NADPH in various strains (C) Total glutathione content in various C. elegans strains in nmole/mg protein. (D) Ratio of GSH:GSSG in different strains. Where N2, wild-type; sin-3;him-5, sin-3(tm1279);him-5(e1490) mutant; sin-3;him-5 Vit. C, sin-3(tm1279);him-5(e1490); him-5(e1490) Vit. C, him-5(e1490) worms maintained on NGM plate supplemented with 10 mM vitamin C. Other mutants are duly indicated on the graph (ns, non-significant; ** P

    Techniques Used: Mutagenesis

    7) Product Images from "IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity"

    Article Title: IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

    Cisplatin exposure of IDH1 MUT HCT116 cells decrease NADPH levels and increase ROS levels, and AGI-5198 attenuates these effects. A ) Cells were incubated in the presence or absence of 1 µM AGI-5198, treated with cisplatin (5 µM), and harvested, prepared, and colorimetrically analyzed for NADP + :NADPH ratios after 72 h. B ) As in A , but cells were treated with cisplatin (25 µM) and analyzed with a fluorometric assay for ROS levels at different time points. * P
    Figure Legend Snippet: Cisplatin exposure of IDH1 MUT HCT116 cells decrease NADPH levels and increase ROS levels, and AGI-5198 attenuates these effects. A ) Cells were incubated in the presence or absence of 1 µM AGI-5198, treated with cisplatin (5 µM), and harvested, prepared, and colorimetrically analyzed for NADP + :NADPH ratios after 72 h. B ) As in A , but cells were treated with cisplatin (25 µM) and analyzed with a fluorometric assay for ROS levels at different time points. * P

    Techniques Used: Incubation

    8) Product Images from "Redesign and reconstruction of a steviol-biosynthetic pathway for enhanced production of steviol in Escherichia coli"

    Article Title: Redesign and reconstruction of a steviol-biosynthetic pathway for enhanced production of steviol in Escherichia coli

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-020-1291-x

    The design of fusion versions of CYP714A2-AtCPR2. The impact of the expression of the fusion proteins on steviol production is illustrated too. a Schematic representation of the design of the three fusion proteins (Fusion 5, Fusion 10, and Fusion 15) with three linker peptides of different lengths (GGGGS) n=1–3 . b The GC–MS spectrum of steviol methyl ester that was generated after methylation of steviol produced by fermentation. c The effect of the expression of three fusion proteins on steviol production by the MGIUKN strain in flask fermentation reactions. The non-fusion form of UtrCYP714A2_AtCPR2 served as a control. The results represent means from five independent experiments. d Homology models of A. thaliana trCYP714A2 fused to trAtCPR2. TrAtCPR2 is white, trCYP714A2 is orange, the linker peptides are red, and NADPH-binding residues are cyan
    Figure Legend Snippet: The design of fusion versions of CYP714A2-AtCPR2. The impact of the expression of the fusion proteins on steviol production is illustrated too. a Schematic representation of the design of the three fusion proteins (Fusion 5, Fusion 10, and Fusion 15) with three linker peptides of different lengths (GGGGS) n=1–3 . b The GC–MS spectrum of steviol methyl ester that was generated after methylation of steviol produced by fermentation. c The effect of the expression of three fusion proteins on steviol production by the MGIUKN strain in flask fermentation reactions. The non-fusion form of UtrCYP714A2_AtCPR2 served as a control. The results represent means from five independent experiments. d Homology models of A. thaliana trCYP714A2 fused to trAtCPR2. TrAtCPR2 is white, trCYP714A2 is orange, the linker peptides are red, and NADPH-binding residues are cyan

    Techniques Used: Expressing, Gas Chromatography-Mass Spectrometry, Generated, Methylation, Produced, Binding Assay

    The biosynthetic pathway of steviol constructed in a heterologous host, E. coli. IPP, the precursor of steviol, is synthesized by the endogenous MEP pathway of E. coli . FPP is converted to steviol by the exogenous steviol synthesis pathway. Single arrows represent single-step reactions, while triple arrows denote multistep reactions. Red arrows indicate overexpressed genes intended to enhance the precursor pool. The genes ( dxs from Bacillus subtilis , dxr from E. coli , and idi and ispA from Enterococcus sp.) were integrated into the genome of the MG1655 strain. CDPS, ent -copalyl diphosphate synthase; CPR, NADPH–cytochrome P450 reductase; DMAPP, dimethylallyl pyrophosphate; DXP, 1-deoxy- d -xylulose 5-phosphate; DXR, 1-deoxy- d -xylulose 5-phosphate reductoisomerase; DXS, 1-deoxyxylulose-5-phosphate synthase; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; GPP, geranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; IspA, farnesyl diphosphate synthase; KAH, kaurenoic acid 13-hydroxylase; KO, ent -kaurene oxidase; KS, ent -kaurene synthase; MEP, 2-C-methylerythritol 4-phosphate; Pyr, pyruvate
    Figure Legend Snippet: The biosynthetic pathway of steviol constructed in a heterologous host, E. coli. IPP, the precursor of steviol, is synthesized by the endogenous MEP pathway of E. coli . FPP is converted to steviol by the exogenous steviol synthesis pathway. Single arrows represent single-step reactions, while triple arrows denote multistep reactions. Red arrows indicate overexpressed genes intended to enhance the precursor pool. The genes ( dxs from Bacillus subtilis , dxr from E. coli , and idi and ispA from Enterococcus sp.) were integrated into the genome of the MG1655 strain. CDPS, ent -copalyl diphosphate synthase; CPR, NADPH–cytochrome P450 reductase; DMAPP, dimethylallyl pyrophosphate; DXP, 1-deoxy- d -xylulose 5-phosphate; DXR, 1-deoxy- d -xylulose 5-phosphate reductoisomerase; DXS, 1-deoxyxylulose-5-phosphate synthase; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; GPP, geranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; IspA, farnesyl diphosphate synthase; KAH, kaurenoic acid 13-hydroxylase; KO, ent -kaurene oxidase; KS, ent -kaurene synthase; MEP, 2-C-methylerythritol 4-phosphate; Pyr, pyruvate

    Techniques Used: Construct, Synthesized

    9) Product Images from "Niacinamide Protects Skin Cells from Oxidative Stress Induced by Particulate Matter"

    Article Title: Niacinamide Protects Skin Cells from Oxidative Stress Induced by Particulate Matter

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2019.061

    Schematic diagram showing the protective action of NIA on PM 2.5 -induced cell damage. NIA protected keratinocytes by suppressing ROS generation by decreasing the NADP/NADPH ratio. Further, NIA prevented oxidative stress-induced molecules damage, including lipid peroxidation, protein carbonylation, and DNA modification. NIA could also stabilize mitochondrial membrane potential by balancing calcium levels, which was disrupted by PM 2.5 . Finally, NIA protected cells from PM 2.5 -induced apoptosis.
    Figure Legend Snippet: Schematic diagram showing the protective action of NIA on PM 2.5 -induced cell damage. NIA protected keratinocytes by suppressing ROS generation by decreasing the NADP/NADPH ratio. Further, NIA prevented oxidative stress-induced molecules damage, including lipid peroxidation, protein carbonylation, and DNA modification. NIA could also stabilize mitochondrial membrane potential by balancing calcium levels, which was disrupted by PM 2.5 . Finally, NIA protected cells from PM 2.5 -induced apoptosis.

    Techniques Used: Modification

    NIA cleared ROS by inhibiting NOX activity induced by PM 2.5 . (A) The ratio of intracellular NADP and NADPH was assessed using NADP/NADPH assay kit. (B) Intracellular ROS was detected after staining of cells with DCF-DA dye. (C) Superoxide generation was detected after dying cells with DHE. NIA diminished superoxide levels induced by PM 2.5 . * p
    Figure Legend Snippet: NIA cleared ROS by inhibiting NOX activity induced by PM 2.5 . (A) The ratio of intracellular NADP and NADPH was assessed using NADP/NADPH assay kit. (B) Intracellular ROS was detected after staining of cells with DCF-DA dye. (C) Superoxide generation was detected after dying cells with DHE. NIA diminished superoxide levels induced by PM 2.5 . * p

    Techniques Used: Activity Assay, Staining

    10) Product Images from "Copper-Induced Activation of MAPKs, CDPKs and CaMKs Triggers Activation of Hexokinase and Inhibition of Pyruvate Kinase Leading to Increased Synthesis of ASC, GSH and NADPH in Ulva compressa"

    Article Title: Copper-Induced Activation of MAPKs, CDPKs and CaMKs Triggers Activation of Hexokinase and Inhibition of Pyruvate Kinase Leading to Increased Synthesis of ASC, GSH and NADPH in Ulva compressa

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.00990

    Scheme of copper-induced increase in ROS and intracellular calcium levels leading to activation of MAPK, CDPKs and CaMKs signaling pathways, which coupled to the increase in net photosynthesis, allow the increase in ASC, GSH and NADPH levels to cope with copper-induced oxidative stress in the marine alga U. compressa . Arrows in blue indicate activation, arrows in red indicate inhibition, the arrow in green indicates extrusion, and choppy arrows indicate results that do not belong to this study.
    Figure Legend Snippet: Scheme of copper-induced increase in ROS and intracellular calcium levels leading to activation of MAPK, CDPKs and CaMKs signaling pathways, which coupled to the increase in net photosynthesis, allow the increase in ASC, GSH and NADPH levels to cope with copper-induced oxidative stress in the marine alga U. compressa . Arrows in blue indicate activation, arrows in red indicate inhibition, the arrow in green indicates extrusion, and choppy arrows indicate results that do not belong to this study.

    Techniques Used: Activation Assay, Inhibition

    Level of total ascorbate (ASC, A ), total glutathione (GSH, B ) and total NADPH ( C ) corresponding to ASC, GSH and NADPH levels (white bars) and dehydroascorbate (DHA), oxidized glutathione (GSSG) and NADP (black bars) in U. compressa cultivated in seawater without copper addition for 5 days, with 10 µM of copper for 5 days, and with 100 µM of W-7, 10 µM staurosporine (St), with 5 µM of PD-98059 (PD) and 10 µM copper for 5 days (hashed bars). Levels of ASC, DHA, GSH, GSSG, NADPH and NADP are expressed in micromoles per gram of fresh tissue (FT). Bar represent mean values of three independent experiments. Different letters indicate significant differences among mean values (±SD) ( P
    Figure Legend Snippet: Level of total ascorbate (ASC, A ), total glutathione (GSH, B ) and total NADPH ( C ) corresponding to ASC, GSH and NADPH levels (white bars) and dehydroascorbate (DHA), oxidized glutathione (GSSG) and NADP (black bars) in U. compressa cultivated in seawater without copper addition for 5 days, with 10 µM of copper for 5 days, and with 100 µM of W-7, 10 µM staurosporine (St), with 5 µM of PD-98059 (PD) and 10 µM copper for 5 days (hashed bars). Levels of ASC, DHA, GSH, GSSG, NADPH and NADP are expressed in micromoles per gram of fresh tissue (FT). Bar represent mean values of three independent experiments. Different letters indicate significant differences among mean values (±SD) ( P

    Techniques Used:

    Related Articles

    Colorimetric Assay:

    Article Title: The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion
    Article Snippet: .. Total nicotinamide adenine dinucleotide levels Extraction and detection of total nicotinamide adenine dinucleotide (NAD) levels was performed using an NAD/NADH colorimetric assay kit (Abcam) according to the manual with minor adaptations. ..

    Nad NADH Assay:

    Article Title: Amyloid-β Increases Tau by Mediating Sirtuin 3 in Alzheimer’s Disease
    Article Snippet: .. Total NAD and NADH were tested using an NAD+ / NADH assay kit (no. ab65348, Abcam, Cambridge, MA) according to the manufacturer’s protocol. ..

    Article Title: Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne syndrome
    Article Snippet: .. Following homogenization with a micro pestle, an NAD/NADH Assay Kit (Abcam, ab65348) was used to quantify NAD+ and NADH levels. .. Audiometry The ABR assay is a quantitative assessment of the neurological response, measured as evoked potential, detected within 10 ms of an auditory stimulus.

    Article Title: An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba
    Article Snippet: .. Measurement of intracellular NAD+ /NADH Intracellular NAD+ and NADH were determined as per the manufacturer's protocol (NAD+ /NADH Assay Kit, Cat No: ab65348, Abcam). .. Briefly 2 × 106 cells were lysed in 400 μl of NAD+ /NADH extraction buffer by sonication (five pulses at 15 amp for 15 s).

    Article Title: The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation
    Article Snippet: .. To measure the NAD/NADH ratio, samples were thawed then processed using the NAD/NADH assay kit (ab65348; Abcam) according to the manufacturer’s instructions. .. Inductively coupled plasma mass spectrometry.

    Article Title: AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia
    Article Snippet: .. Quantification of NADH and NAD, and their ratio was carried out with a colorimetric NAD/NADH assay kit (Abcam), according to the manufacturer’s instructions. .. RNA interference Small interfering RNA (siRNA) targeted at mouse AMPK (Santa Cruz Biotechnology) was used to knockout AMPK.

    other:

    Article Title: Sirtuin 3 mediates neuroprotection of ketones against ischemic stroke
    Article Snippet: NAD+ , NADH, and their ratio were detected by using the NADH/NAD Quantification Kit (ab65348, Abcam, Cambridge, MA, USA) according to the manufacturer's protocol.

    Homogenization:

    Article Title: Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne syndrome
    Article Snippet: .. Following homogenization with a micro pestle, an NAD/NADH Assay Kit (Abcam, ab65348) was used to quantify NAD+ and NADH levels. .. Audiometry The ABR assay is a quantitative assessment of the neurological response, measured as evoked potential, detected within 10 ms of an auditory stimulus.

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  • 93
    Abcam nadph level
    Schema indicating the role of <t>TGFβ1-FOXM1-HMGA1-G6PD</t> axis in cisplatin resistance of NSCLC. TGFβ1 stimulation prevents the ubiquitination and degradation of FOXM1 protein, a transcription factor for HMGA1 gene, thereby activating the transcription of HMGA1. Upregulated HMGA1 further activates the transcription of G6PD to promote the PPP, which supplies <t>NADPH</t> and dNTP against the ROS and DNA damage caused by cisplatin. Moreover, HMGA1 induces the production and secretion of TGFβ1, which in turn enhances TGFβ1 signaling to maintain G6PD expression and chemoresistance.
    Nadph Level, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam nadp icdh
    Comparison of the relationship between temperature and E o for respiration in intact spadices and mitochondria in skunk cabbage. ( a ) Curve fitting of the respiration rates in intact spadices using a modified Arrhenius model. Data are derived from Seymour et al . 7 . ( b ) Curve fitting of the respiration rates in isolated mitochondria using a modified Arrhenius model. Data are from mitochondrial respiration operated by <t>NADP</t> + <t>-ICDH</t> and NDA (NADPH-NDA/ICDH; green), and by NAD + -NDB (NADH-NDB; light blue). Respiration rates were determined under constant temperature at 8 °C, 15 °C, 23 °C or 30 °C in respiration via NADPH-NDA/ICDH and at 15 °C, 23 °C, 30 °C or 37 °C via NADH-NDB (n = 6). ( c ) Determination of temperature responses of E o for intact spadices and mitochondrial respiration. Changes of E o value for intact spadices (red), isolated mitochondria (NADPH-NDA/ICDH (green) and NADH-NDB (light blue)) are depicted (n = 6). Intersection points of E o are shown for spadices and NADPH-NDA/ICDH at 15.2 °C and 22.3 °C, respectively.
    Nadp Icdh, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Schema indicating the role of TGFβ1-FOXM1-HMGA1-G6PD axis in cisplatin resistance of NSCLC. TGFβ1 stimulation prevents the ubiquitination and degradation of FOXM1 protein, a transcription factor for HMGA1 gene, thereby activating the transcription of HMGA1. Upregulated HMGA1 further activates the transcription of G6PD to promote the PPP, which supplies NADPH and dNTP against the ROS and DNA damage caused by cisplatin. Moreover, HMGA1 induces the production and secretion of TGFβ1, which in turn enhances TGFβ1 signaling to maintain G6PD expression and chemoresistance.

    Journal: American Journal of Translational Research

    Article Title: The TGFβ1-FOXM1-HMGA1-TGFβ1 positive feedback loop increases the cisplatin resistance of non-small cell lung cancer by inducing G6PD expression

    doi:

    Figure Lengend Snippet: Schema indicating the role of TGFβ1-FOXM1-HMGA1-G6PD axis in cisplatin resistance of NSCLC. TGFβ1 stimulation prevents the ubiquitination and degradation of FOXM1 protein, a transcription factor for HMGA1 gene, thereby activating the transcription of HMGA1. Upregulated HMGA1 further activates the transcription of G6PD to promote the PPP, which supplies NADPH and dNTP against the ROS and DNA damage caused by cisplatin. Moreover, HMGA1 induces the production and secretion of TGFβ1, which in turn enhances TGFβ1 signaling to maintain G6PD expression and chemoresistance.

    Article Snippet: Following transfection and treatment, G6PD activity, NADPH level, and reactive oxygen species (ROS) level were tested using the Glucose 6 Phosphate Dehydrogenase Assay Kit (ab102529; abcam), NADP/NADPH Assay Kit (ab65349; abcam), and ROS/Superoxide Detection Assay Kit (ab139476; abcam) according to each manufacturer’s specifications.

    Techniques: Expressing

    Comparison of the relationship between temperature and E o for respiration in intact spadices and mitochondria in skunk cabbage. ( a ) Curve fitting of the respiration rates in intact spadices using a modified Arrhenius model. Data are derived from Seymour et al . 7 . ( b ) Curve fitting of the respiration rates in isolated mitochondria using a modified Arrhenius model. Data are from mitochondrial respiration operated by NADP + -ICDH and NDA (NADPH-NDA/ICDH; green), and by NAD + -NDB (NADH-NDB; light blue). Respiration rates were determined under constant temperature at 8 °C, 15 °C, 23 °C or 30 °C in respiration via NADPH-NDA/ICDH and at 15 °C, 23 °C, 30 °C or 37 °C via NADH-NDB (n = 6). ( c ) Determination of temperature responses of E o for intact spadices and mitochondrial respiration. Changes of E o value for intact spadices (red), isolated mitochondria (NADPH-NDA/ICDH (green) and NADH-NDB (light blue)) are depicted (n = 6). Intersection points of E o are shown for spadices and NADPH-NDA/ICDH at 15.2 °C and 22.3 °C, respectively.

    Journal: Scientific Reports

    Article Title: The biochemical basis for thermoregulation in heat-producing flowers

    doi: 10.1038/srep24830

    Figure Lengend Snippet: Comparison of the relationship between temperature and E o for respiration in intact spadices and mitochondria in skunk cabbage. ( a ) Curve fitting of the respiration rates in intact spadices using a modified Arrhenius model. Data are derived from Seymour et al . 7 . ( b ) Curve fitting of the respiration rates in isolated mitochondria using a modified Arrhenius model. Data are from mitochondrial respiration operated by NADP + -ICDH and NDA (NADPH-NDA/ICDH; green), and by NAD + -NDB (NADH-NDB; light blue). Respiration rates were determined under constant temperature at 8 °C, 15 °C, 23 °C or 30 °C in respiration via NADPH-NDA/ICDH and at 15 °C, 23 °C, 30 °C or 37 °C via NADH-NDB (n = 6). ( c ) Determination of temperature responses of E o for intact spadices and mitochondrial respiration. Changes of E o value for intact spadices (red), isolated mitochondria (NADPH-NDA/ICDH (green) and NADH-NDB (light blue)) are depicted (n = 6). Intersection points of E o are shown for spadices and NADPH-NDA/ICDH at 15.2 °C and 22.3 °C, respectively.

    Article Snippet: Enzyme assays Enzymatic activities for mitochondrial aconitase and NAD+ - or NADP+ -ICDH were determined using an Aconitase Assay Kit (Abcam) and an Isocitrate Dehydrogenase Assay Kit (Abcam), respectively, and at 15 °C, 23 °C and 30 °C.

    Techniques: Modification, Derivative Assay, Isolation

    Effects of pyruvate on the dynamic temperature response ( δ ) of E o in isolated mitochondria. Values for δ of NADPH-NDA/ICDH- and NADH-NDB-mediated oxygen consumptions for AOX capacities were determined in the absence (−) or presence (+) of pyruvate (n = 3). n.s.: not significant.

    Journal: Scientific Reports

    Article Title: The biochemical basis for thermoregulation in heat-producing flowers

    doi: 10.1038/srep24830

    Figure Lengend Snippet: Effects of pyruvate on the dynamic temperature response ( δ ) of E o in isolated mitochondria. Values for δ of NADPH-NDA/ICDH- and NADH-NDB-mediated oxygen consumptions for AOX capacities were determined in the absence (−) or presence (+) of pyruvate (n = 3). n.s.: not significant.

    Article Snippet: Enzyme assays Enzymatic activities for mitochondrial aconitase and NAD+ - or NADP+ -ICDH were determined using an Aconitase Assay Kit (Abcam) and an Isocitrate Dehydrogenase Assay Kit (Abcam), respectively, and at 15 °C, 23 °C and 30 °C.

    Techniques: Isolation

    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