Structured Review

Abcam nadp nadph
Cancer energy asset. Panel ( A ) represents the function of mitochondrial respiratory Complex I in CT26 and 4T1 cell lines, respectively. MTF virtually abolished Complex I activity (expressed as mU x mg −1 of proteins) that was instead left unaltered by all remaining treatments. This effect resulted in a marked decrease in the rate of both oxygen consumption (expressed as μMol O 2 x min −1 x mg −1 or proteins in Panel ( B ) and ATP synthesis (expressed as nMol x min −1 x mg −1 or proteins in Panel ( C ) through the pathway I-III-IV interrogated by pyruvate-malate administration. Despite this markedly different effect on OXPHOS, ATP:AMP ratio was significantly decreased also by CBX and siRNA (Panel D ) though to a lower degree with respect to MTF. Panels ( E , F ) display the original gels, run under the same experimental conditions for each cell line, documenting the expected response of the energy sensor mechanism that caused an increase in p-AMPK without altering total AMPK levels. The redox nature of H6PD triggered metabolism was confirmed by the decrease in NAD + availability, since NAD + /NADH ratio was selectively decreased by MTF (panel G ). By contrast, lactate release (expressed as mMol/10 6 cells over 24 hours) was induced by all interventions but scramble (Panel H ) despite an absent response of NADH levels. On the contrary, both CBX and siRNA, differently from the biguanide, increased the <t>NADP</t> + <t>/NADPH</t> ratio, without altering total coenzyme levels (measured in picoMol/10 6 cells) (Panels I , J ). (*=p
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1) Product Images from "Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt"

Article Title: Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

Journal: Scientific Reports

doi: 10.1038/srep25092

Cancer energy asset. Panel ( A ) represents the function of mitochondrial respiratory Complex I in CT26 and 4T1 cell lines, respectively. MTF virtually abolished Complex I activity (expressed as mU x mg −1 of proteins) that was instead left unaltered by all remaining treatments. This effect resulted in a marked decrease in the rate of both oxygen consumption (expressed as μMol O 2 x min −1 x mg −1 or proteins in Panel ( B ) and ATP synthesis (expressed as nMol x min −1 x mg −1 or proteins in Panel ( C ) through the pathway I-III-IV interrogated by pyruvate-malate administration. Despite this markedly different effect on OXPHOS, ATP:AMP ratio was significantly decreased also by CBX and siRNA (Panel D ) though to a lower degree with respect to MTF. Panels ( E , F ) display the original gels, run under the same experimental conditions for each cell line, documenting the expected response of the energy sensor mechanism that caused an increase in p-AMPK without altering total AMPK levels. The redox nature of H6PD triggered metabolism was confirmed by the decrease in NAD + availability, since NAD + /NADH ratio was selectively decreased by MTF (panel G ). By contrast, lactate release (expressed as mMol/10 6 cells over 24 hours) was induced by all interventions but scramble (Panel H ) despite an absent response of NADH levels. On the contrary, both CBX and siRNA, differently from the biguanide, increased the NADP + /NADPH ratio, without altering total coenzyme levels (measured in picoMol/10 6 cells) (Panels I , J ). (*=p
Figure Legend Snippet: Cancer energy asset. Panel ( A ) represents the function of mitochondrial respiratory Complex I in CT26 and 4T1 cell lines, respectively. MTF virtually abolished Complex I activity (expressed as mU x mg −1 of proteins) that was instead left unaltered by all remaining treatments. This effect resulted in a marked decrease in the rate of both oxygen consumption (expressed as μMol O 2 x min −1 x mg −1 or proteins in Panel ( B ) and ATP synthesis (expressed as nMol x min −1 x mg −1 or proteins in Panel ( C ) through the pathway I-III-IV interrogated by pyruvate-malate administration. Despite this markedly different effect on OXPHOS, ATP:AMP ratio was significantly decreased also by CBX and siRNA (Panel D ) though to a lower degree with respect to MTF. Panels ( E , F ) display the original gels, run under the same experimental conditions for each cell line, documenting the expected response of the energy sensor mechanism that caused an increase in p-AMPK without altering total AMPK levels. The redox nature of H6PD triggered metabolism was confirmed by the decrease in NAD + availability, since NAD + /NADH ratio was selectively decreased by MTF (panel G ). By contrast, lactate release (expressed as mMol/10 6 cells over 24 hours) was induced by all interventions but scramble (Panel H ) despite an absent response of NADH levels. On the contrary, both CBX and siRNA, differently from the biguanide, increased the NADP + /NADPH ratio, without altering total coenzyme levels (measured in picoMol/10 6 cells) (Panels I , J ). (*=p

Techniques Used: Activity Assay

2) Product Images from "PFKFB3 regulates oxidative stress homeostasis via its S-glutathionylation in cancer"

Article Title: PFKFB3 regulates oxidative stress homeostasis via its S-glutathionylation in cancer

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2013.11.021

PFKFB3 S-glutathionylation increases the carbohydrate flux to the PPP ( a ) Intracellular Fru-2,6-P 2 concentration, ( b ) the secreted lactate levels ( c ), cellular concentration of Ribul-5P and Xyl-5P, ( d ) NADPH, and ( e ) GSH/GSSG ratio in HeLa cells transiently expressing empty vector pcDNA3.1 (Con), expression plasmids for WT- or C206A-PFKFB3 after treatment with 100 μM H 2 O 2 , 50 μM BCNU, or 1.5 mM GSHee to induce PFKFB3 S-glutathionylation. Absolute values were normalized to the total protein concentration and changes in metabolite levels are expressed as a percent of control (without oxidant treatment). T-test (*P
Figure Legend Snippet: PFKFB3 S-glutathionylation increases the carbohydrate flux to the PPP ( a ) Intracellular Fru-2,6-P 2 concentration, ( b ) the secreted lactate levels ( c ), cellular concentration of Ribul-5P and Xyl-5P, ( d ) NADPH, and ( e ) GSH/GSSG ratio in HeLa cells transiently expressing empty vector pcDNA3.1 (Con), expression plasmids for WT- or C206A-PFKFB3 after treatment with 100 μM H 2 O 2 , 50 μM BCNU, or 1.5 mM GSHee to induce PFKFB3 S-glutathionylation. Absolute values were normalized to the total protein concentration and changes in metabolite levels are expressed as a percent of control (without oxidant treatment). T-test (*P

Techniques Used: Concentration Assay, Expressing, Plasmid Preparation, Protein Concentration

A model for the carbohydrate flux upon PFKFB3 S-glutathionylation ( a ) This diagram is provided to shows a hypothetical role played by S-glutathionylation of PFKFB3 in a relationship between fructose-2,6-bisphosphate (Fru-2,6-BP), PFKFB3, and the PPP. It has been hypothesized that increased S-glutathionylation of PFKFB3 leads to decreases in the Fru-2,6-BP level and in the carbohydrate flux to glycolysis and, in turn, an increase in the flux to the PPP and the level of GSH. Solid/bold lines represent direct, one-step biochemical reactions, and indirect, multi-step reactions are represented by dotted lines. Inhibition and activation of enzymatic steps are indicated by solid/plain lines. G-6-P, glucose-6-phosphate; Ribul-5-P, ribulose-5-phosphate; Fru-6-P, fructose -6-phosphate; Fru-1,6-BP, fructose-1,6-bisphosphate; Fru-2,6-BP, fructose-2,6-bisphosphate; PFKFB3-SSG, S-glutathionylated PFKFB3; PFKFB3-SH, unglutathionylated PFKFB3. ( b ) Enzymatic assay for rerouting of carbohydrate metabolic flux in response to PFKFB3 S-glutathionylation. Changes in the formation of Fru-2,6-BP and NADPH from varied extents of S-glutathionylation of PFKFB3 was measured. The extent of S-glutathionylation was controlled with varied molar ratio of GSH to GSSG in a total concentration kept as 3 mM.
Figure Legend Snippet: A model for the carbohydrate flux upon PFKFB3 S-glutathionylation ( a ) This diagram is provided to shows a hypothetical role played by S-glutathionylation of PFKFB3 in a relationship between fructose-2,6-bisphosphate (Fru-2,6-BP), PFKFB3, and the PPP. It has been hypothesized that increased S-glutathionylation of PFKFB3 leads to decreases in the Fru-2,6-BP level and in the carbohydrate flux to glycolysis and, in turn, an increase in the flux to the PPP and the level of GSH. Solid/bold lines represent direct, one-step biochemical reactions, and indirect, multi-step reactions are represented by dotted lines. Inhibition and activation of enzymatic steps are indicated by solid/plain lines. G-6-P, glucose-6-phosphate; Ribul-5-P, ribulose-5-phosphate; Fru-6-P, fructose -6-phosphate; Fru-1,6-BP, fructose-1,6-bisphosphate; Fru-2,6-BP, fructose-2,6-bisphosphate; PFKFB3-SSG, S-glutathionylated PFKFB3; PFKFB3-SH, unglutathionylated PFKFB3. ( b ) Enzymatic assay for rerouting of carbohydrate metabolic flux in response to PFKFB3 S-glutathionylation. Changes in the formation of Fru-2,6-BP and NADPH from varied extents of S-glutathionylation of PFKFB3 was measured. The extent of S-glutathionylation was controlled with varied molar ratio of GSH to GSSG in a total concentration kept as 3 mM.

Techniques Used: Inhibition, Activation Assay, Enzymatic Assay, Concentration Assay

3) Product Images from "NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic ?-cells"

Article Title: NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic ?-cells

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00465.2011

NADK Overexpression and its Effect on NADPH levels, the NADPH/NADP+ Ratio, and Insulin Secretion
Figure Legend Snippet: NADK Overexpression and its Effect on NADPH levels, the NADPH/NADP+ Ratio, and Insulin Secretion

Techniques Used: Over Expression

Effect of NADK overexpression knockdown on total (NADPH + NADP + ) levels ( A ) and insulin secretion ( B ) in mouse islets. Nucleotide determination and insulin secretion were performed as described in the legend to . Data are means ± SE from
Figure Legend Snippet: Effect of NADK overexpression knockdown on total (NADPH + NADP + ) levels ( A ) and insulin secretion ( B ) in mouse islets. Nucleotide determination and insulin secretion were performed as described in the legend to . Data are means ± SE from

Techniques Used: Over Expression

Effect of NADK overexpression on NADK mRNA ( A ), total (NADPH + NADP + ) levels ( B ), NADPH/(NADPH + NADP + ) ratio ( C ), insulin secretion ( D ), and NADH/(NADH + NAD + ) and ATP/ADP ratio ( E ) in INS-1 832/13 cells. Nucleotides were determined using NADPH/NADP
Figure Legend Snippet: Effect of NADK overexpression on NADK mRNA ( A ), total (NADPH + NADP + ) levels ( B ), NADPH/(NADPH + NADP + ) ratio ( C ), insulin secretion ( D ), and NADH/(NADH + NAD + ) and ATP/ADP ratio ( E ) in INS-1 832/13 cells. Nucleotides were determined using NADPH/NADP

Techniques Used: Over Expression

Effect of NADK knockdown on NADK mRNA ( A ), total (NADPH + NADP + ) levels ( B ), the NADPH/(NADPH + NADP + ) ratio ( C ), insulin secretion ( D ), and NADH/(NADH + NAD + ) and ATP/ADP ratio ( E ) in INS-1 832/13 cells. Nucleotide determination and insulin secretion
Figure Legend Snippet: Effect of NADK knockdown on NADK mRNA ( A ), total (NADPH + NADP + ) levels ( B ), the NADPH/(NADPH + NADP + ) ratio ( C ), insulin secretion ( D ), and NADH/(NADH + NAD + ) and ATP/ADP ratio ( E ) in INS-1 832/13 cells. Nucleotide determination and insulin secretion

Techniques Used:

4) Product Images from "Alterations in fatty acid metabolism and sirtuin signaling characterize early type‐2 diabetic hearts of fructose‐fed rats. Alterations in fatty acid metabolism and sirtuin signaling characterize early type‐2 diabetic hearts of fructose‐fed rats"

Article Title: Alterations in fatty acid metabolism and sirtuin signaling characterize early type‐2 diabetic hearts of fructose‐fed rats. Alterations in fatty acid metabolism and sirtuin signaling characterize early type‐2 diabetic hearts of fructose‐fed rats

Journal: Physiological Reports

doi: 10.14814/phy2.13388

Increased oxidative stress in hearts of fructose‐fed rats. (A) Representative immunoblots and densitometric quantification of mitochondrial 4‐hydroxynonenal ( HNE ), mitochondrial thioredoxin reductase (TrxR2), and cellular glutathione peroxidase 1 ( GP x1) ( n = 6–7/group). (B) Concentrations of reduced glutathione ( GSH ) and oxidized glutathione ( GSSG ) in cardiac homogenates of control and fructose‐fed rats, with their respective GSH : GSSG ratio ( n = 10/group). (C) Concentrations of NADPH and NADP + in cardiac mitochondria of control and fructose‐fed rats, with their respective NADPH / NADP + ratio ( n = 8/group). Data are presented as mean ± SE. # Mann–Whitney nonparametric statistical test was applied. C, control rats; FF , fructose‐fed rats.
Figure Legend Snippet: Increased oxidative stress in hearts of fructose‐fed rats. (A) Representative immunoblots and densitometric quantification of mitochondrial 4‐hydroxynonenal ( HNE ), mitochondrial thioredoxin reductase (TrxR2), and cellular glutathione peroxidase 1 ( GP x1) ( n = 6–7/group). (B) Concentrations of reduced glutathione ( GSH ) and oxidized glutathione ( GSSG ) in cardiac homogenates of control and fructose‐fed rats, with their respective GSH : GSSG ratio ( n = 10/group). (C) Concentrations of NADPH and NADP + in cardiac mitochondria of control and fructose‐fed rats, with their respective NADPH / NADP + ratio ( n = 8/group). Data are presented as mean ± SE. # Mann–Whitney nonparametric statistical test was applied. C, control rats; FF , fructose‐fed rats.

Techniques Used: Western Blot, MANN-WHITNEY

5) Product Images from "Mechanisms of Doxorubicin Toxicity in Pancreatic β-Cells"

Article Title: Mechanisms of Doxorubicin Toxicity in Pancreatic β-Cells

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfw096

Doxorubicin causes a significant dose- and time-dependent reduction in the total NADH/NAD + pool and ATP content. INS-1 832/13 cells were treated with doxorubicin for 1 h. Following doxorubicin removal and 6 h recovery in growth media, cells were analyzed for (A) (NADPH + NADP + ) and (NADH + NAD + ) Asterisks indicate a significant difference ( P
Figure Legend Snippet: Doxorubicin causes a significant dose- and time-dependent reduction in the total NADH/NAD + pool and ATP content. INS-1 832/13 cells were treated with doxorubicin for 1 h. Following doxorubicin removal and 6 h recovery in growth media, cells were analyzed for (A) (NADPH + NADP + ) and (NADH + NAD + ) Asterisks indicate a significant difference ( P

Techniques Used:

6) Product Images from "OGDH promotes the progression of gastric cancer by regulating mitochondrial bioenergetics and Wnt/β-catenin signal pathway"

Article Title: OGDH promotes the progression of gastric cancer by regulating mitochondrial bioenergetics and Wnt/β-catenin signal pathway

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S208848

OGDH augments mitochondrial functions. ( A ) OGDH siRNA inhibits mitochondrial membrance potential (ΔΨm), intracellular ATP concentration and O2 consumption rate in AGS cells. ( B ) Ectopic expression of OGDH increases mitochondrial bioenergetics in BGC823 cells. ( C ) OGDH siRNA upregulates ROS level and NADP + /NADPH ratio. ( D ) The levels of ROS and NADP + /NADPH ratio are downregulated by OGDH overexpression. Values are presented as mean ± SEM from 3 independent cultures. * P
Figure Legend Snippet: OGDH augments mitochondrial functions. ( A ) OGDH siRNA inhibits mitochondrial membrance potential (ΔΨm), intracellular ATP concentration and O2 consumption rate in AGS cells. ( B ) Ectopic expression of OGDH increases mitochondrial bioenergetics in BGC823 cells. ( C ) OGDH siRNA upregulates ROS level and NADP + /NADPH ratio. ( D ) The levels of ROS and NADP + /NADPH ratio are downregulated by OGDH overexpression. Values are presented as mean ± SEM from 3 independent cultures. * P

Techniques Used: Concentration Assay, Expressing, Over Expression

7) Product Images from "Thymoquinone, a bioactive component of Nigella sativa, normalizes insulin secretion from pancreatic β-cells under glucose overload via regulation of malonyl-CoA"

Article Title: Thymoquinone, a bioactive component of Nigella sativa, normalizes insulin secretion from pancreatic β-cells under glucose overload via regulation of malonyl-CoA

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00250.2015

Acute effect of TQ and NSE on NADH/(NADH + NAD + ) and NADPH/(NADPH + NADP + ) ratios in INS-1 832/13 cells. A and B : examples of the TQ ( A ) and NSE (4.45% TQ; B ) effect on the real-time NAD(P)H autofluorescence in a population of INS-1 832/13 cells. C – F : traces are representatives of 3–5 independent experiments. Effect of TQ (5 μM) and NSE (0.1 μg/ml) on the NADH/NAD + ( C and D ) and NADPH/NADP + ratios ( E and F ) were determined following a 1-h exposure of cells to 3 or 16 mM G in the absence or presence of these agents. Data are means ± SE from 2 independent experiments performed in duplicate measurements. * P
Figure Legend Snippet: Acute effect of TQ and NSE on NADH/(NADH + NAD + ) and NADPH/(NADPH + NADP + ) ratios in INS-1 832/13 cells. A and B : examples of the TQ ( A ) and NSE (4.45% TQ; B ) effect on the real-time NAD(P)H autofluorescence in a population of INS-1 832/13 cells. C – F : traces are representatives of 3–5 independent experiments. Effect of TQ (5 μM) and NSE (0.1 μg/ml) on the NADH/NAD + ( C and D ) and NADPH/NADP + ratios ( E and F ) were determined following a 1-h exposure of cells to 3 or 16 mM G in the absence or presence of these agents. Data are means ± SE from 2 independent experiments performed in duplicate measurements. * P

Techniques Used:

Related Articles

Nad NADH Assay:

Article Title: Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt
Article Snippet: .. NAD+ /NADH and NADP/NADPH determination The ratio between NAD+ :NADH and NADP:NADPH in cell lysates were evaluated spectrophotometrically, at 450 nm, using the NAD/NADH Assay Kit (Abcam: ab65348) and NADP/NADPH Assay Kit (Abcam: ab65349), respectively following the manufacture’s instructions. .. Transfection assay Silencing of H6PD expression was achieved by transfecting cells with H6PDH siRNA (Ambion siRNA ID 14371) or SilencerTM Negative Control #1 siRNA (Ambion, Huntingdon, Cambs, UK) both 40 pMoles/ml, using Lipofectamine 2000 as transfection agent (2.5 microL/mL).

other:

Article Title: OGDH promotes the progression of gastric cancer by regulating mitochondrial bioenergetics and Wnt/β-catenin signal pathway
Article Snippet: Measurement of the ratio of NADP+ /NADPH The intracellular ratio NADP+ /NADPH was detected by NADP+ /NADPH Assay kit (#ab65349, Abcam) according to the manufacturer’s instructions.

Article Title: Restriction of Aerobic Metabolism by Acquired or Innate Arylsulfatase B Deficiency: A New Approach to the Warburg Effect
Article Snippet: Total NADP+ and NADPH and NADPH were measured in the hepatic cell and tissue samples by a commercial assay (Abcam).

Article Title: Mechanisms of Doxorubicin Toxicity in Pancreatic β-Cells
Article Snippet: NAD(P)+ , NAD(P)H, and ATP were determined using the NAD+ /NADH, NADP+ /NADPH, and ATP kits (Abcam, Cambridge, Massachusetts) according to the protocols of the manufacturer.

Article Title: PCK1 Downregulation Promotes TXNRD1 Expression and Hepatoma Cell Growth via the Nrf2/Keap1 Pathway
Article Snippet: Measurement of NADP/NADPH NADPH and NADP+ levels were measured using a NADP/NADPH Assay Kit (ab65349, Abcam, Cambridge, UK) according to the manufacturer's instructions.

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    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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    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