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  • 94
    Millipore nadph
    Identification of AmISY. a , phylogenetic tree of iridoid synthase homologues in A. majus ( Am ), Olea europaea ( Oe ), C. roseus ( Cr ), Nepeta cataria ( Nc ), and N. mussinii ( Nm ). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 ( 24 ). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font . One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b , SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c , the <t>8-oxogeranial–dependent</t> <t>NADPH</t> consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μ m (AmISY: k cat = 0.72 ± 0.02 s −1 , K m = 1.1 ± 0.1 μ m ; CrISY: k cat = 1.6 ± 0.1 s −1 , K m = 4.5 ± 0.2 μ m ). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d , qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus . Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.
    Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nadp nadph nadph
    PCK1 suppresses oxidative stress in liver cancer cells. Relative ratios of <t>NADPH</t> to total <t>NADP</t> 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
    Nadp Nadph Nadph, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nadp nadp and nadph
    In-vitro analysis on influence of metal oxides on <t>NADPH</t> regeneration (conditions: cultivation time: 20 days; CuO (2 µM), Fe 2 O 3 (20 µM), MgO (200 µM), MnO (20 µM), MoO 3 (2 µM) and ZnO (2 µM) (a), and light intensity on Fe 2 O 3 (20 µM) and MgO (200 µM) mediated NADPH regeneration (b).
    Nadp Nadp And Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore nadph assay nadph
    6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances the resistance of tumor cells to ionizing radiation (IR). a , b U87/EGFRvIII cells stably expressing luciferase were depleted of endogenous 6PGD and reconstituted with the expression of r6PGD WT or Y481F. These cells were treated with or without X-ray radiation (10 Gy) and cultured for 8 h. Reactive oxygen species (ROS) levels were measured using ROS assay kit ( a ). <t>NADPH/NADP</t> + ratio was determined using NADP + /NADPH quantitation kit ( b ). Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p
    Nadph Assay Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nadp  (Abcam)
    97
    Abcam nadp
    Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced <t>NADP</t> and <t>NAD</t> ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P
    Nadp, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nadp nadph assay nadp nadph levels
    mTOR inhibition reduces NOX expression and antioxidants block oxLDL priming. Cells were treated with 100 nM Torin1 or vehicle an hour before treatment with 20 μg/ml oxLDL or vehicle. On day 3 cells were harvested and mRNA expression of NOX2 (A) and NOX4 (B) was analyzed using real-time qPCR. (C) Cells were treated as described above. Concentrations of <t>NADP</t> and <t>NADPH</t> were measured using a colorimetric assay kit on day 6. Ratio of NADP/NADPH is shown. (D,E) Monocytes were pre-incubated for 1 h with 0.5 μM Diphenyleneiodonium (DPI), 25 μM VAS2870, 40 μM Mito-TEMPO or vehicle before treating with oxLDL or vehicle for 24 h. On day 6 cells were stimulated with Pam3cys and IL6 (D) and TNFα (E) were measured in the supernatant using ELISA. Graphs represent mean values ± SD of at least 6 individuals in at least 3 different experiments. * P
    Nadp Nadph Assay Nadp Nadph Levels, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nicotinamide adenine dinucleotide phosphate nadph
    Increased HO-1 expression leads to inhibition of <t>PDGF-stimulated</t> <t>NADPH</t> oxidase activity
    Nicotinamide Adenine Dinucleotide Phosphate Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Identification of AmISY. a , phylogenetic tree of iridoid synthase homologues in A. majus ( Am ), Olea europaea ( Oe ), C. roseus ( Cr ), Nepeta cataria ( Nc ), and N. mussinii ( Nm ). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 ( 24 ). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font . One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b , SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c , the 8-oxogeranial–dependent NADPH consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μ m (AmISY: k cat = 0.72 ± 0.02 s −1 , K m = 1.1 ± 0.1 μ m ; CrISY: k cat = 1.6 ± 0.1 s −1 , K m = 4.5 ± 0.2 μ m ). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d , qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus . Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.

    Journal: The Journal of Biological Chemistry

    Article Title: Inverted stereocontrol of iridoid synthase in snapdragon

    doi: 10.1074/jbc.M117.800979

    Figure Lengend Snippet: Identification of AmISY. a , phylogenetic tree of iridoid synthase homologues in A. majus ( Am ), Olea europaea ( Oe ), C. roseus ( Cr ), Nepeta cataria ( Nc ), and N. mussinii ( Nm ). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 ( 24 ). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font . One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b , SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c , the 8-oxogeranial–dependent NADPH consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μ m (AmISY: k cat = 0.72 ± 0.02 s −1 , K m = 1.1 ± 0.1 μ m ; CrISY: k cat = 1.6 ± 0.1 s −1 , K m = 4.5 ± 0.2 μ m ). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d , qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus . Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.

    Article Snippet: Reactions were conducted in plastic cuvettes with 1-cm path length and contained 20 nm AmISY in buffer C (200 mm MOPS, pH 7.0, and 100 mm NaCl), 50 μm NADPH (Sigma, N7505), 0.66–5 μm 8-oxogeranial substrate, and 1% THF in a total volume of 800 μl.

    Techniques: Activity Assay, SDS Page, Filtration, Chromatography, Purification, Concentration Assay, Quantitative RT-PCR, Expressing

    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

    Journal: Frontiers in Oncology

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

    doi: 10.3389/fonc.2018.00611

    Figure Lengend 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

    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.

    Techniques: Labeling, Staining, Fluorescence, Infection

    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

    Journal: Scientific Reports

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

    doi: 10.1038/srep25092

    Figure Lengend 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

    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.

    Techniques: Activity Assay

    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.

    Journal: Biomolecules & Therapeutics

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

    doi: 10.4062/biomolther.2019.061

    Figure Lengend 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.

    Article Snippet: NADP/NADPH assay To determine the ratio of intracellular NADP and NADPH, we used NADP/NADPH assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

    Techniques: 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

    Journal: Biomolecules & Therapeutics

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

    doi: 10.4062/biomolther.2019.061

    Figure Lengend 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

    Article Snippet: NADP/NADPH assay To determine the ratio of intracellular NADP and NADPH, we used NADP/NADPH assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Staining

    Effects of SC-66 on glucose metabolism. (A–B) C33A cells were seeded in T 25 cm 2 tissue culture flasks, treated with SC-66 and intracellular NADPH levels and ATP were determined. The graph represents ATP and NADPH µM levels based on standard curve, p

    Journal: PLoS ONE

    Article Title: AKT Inhibitors Promote Cell Death in Cervical Cancer through Disruption of mTOR Signaling and Glucose Uptake

    doi: 10.1371/journal.pone.0092948

    Figure Lengend Snippet: Effects of SC-66 on glucose metabolism. (A–B) C33A cells were seeded in T 25 cm 2 tissue culture flasks, treated with SC-66 and intracellular NADPH levels and ATP were determined. The graph represents ATP and NADPH µM levels based on standard curve, p

    Article Snippet: ATP and NADP/NADPH assays were performed using the commercially available fluorometric kits from (Abcam, Cambridge, MA) according to the manufacturer's instructions using cell lysates.

    Techniques:

    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

    Journal: OncoTargets and therapy

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

    doi: 10.2147/OTT.S208848

    Figure Lengend 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

    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.

    Techniques: Concentration Assay, Expressing, Over Expression

    In-vitro analysis on influence of metal oxides on NADPH regeneration (conditions: cultivation time: 20 days; CuO (2 µM), Fe 2 O 3 (20 µM), MgO (200 µM), MnO (20 µM), MoO 3 (2 µM) and ZnO (2 µM) (a), and light intensity on Fe 2 O 3 (20 µM) and MgO (200 µM) mediated NADPH regeneration (b).

    Journal: bioRxiv

    Article Title: Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by Engineered Synechocystis sp. PCC 6803

    doi: 10.1101/529420

    Figure Lengend Snippet: In-vitro analysis on influence of metal oxides on NADPH regeneration (conditions: cultivation time: 20 days; CuO (2 µM), Fe 2 O 3 (20 µM), MgO (200 µM), MnO (20 µM), MoO 3 (2 µM) and ZnO (2 µM) (a), and light intensity on Fe 2 O 3 (20 µM) and MgO (200 µM) mediated NADPH regeneration (b).

    Article Snippet: The filtered samples were used for total NADP (NADP and NADPH) and heated samples (60°C for 30 min) were used for NADPH analysis.

    Techniques: In Vitro

    Effect of light intensity on biomass (a) and chlorophyll a (b) concentrations (conditions: cultivation time: 20 days; NADP: 150 µM; Fe 2 O 3 : 20 µM and MgO: 200 µM).

    Journal: bioRxiv

    Article Title: Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by Engineered Synechocystis sp. PCC 6803

    doi: 10.1101/529420

    Figure Lengend Snippet: Effect of light intensity on biomass (a) and chlorophyll a (b) concentrations (conditions: cultivation time: 20 days; NADP: 150 µM; Fe 2 O 3 : 20 µM and MgO: 200 µM).

    Article Snippet: The filtered samples were used for total NADP (NADP and NADPH) and heated samples (60°C for 30 min) were used for NADPH analysis.

    Techniques:

    Effect of incubation time on ethanol and biomass concentration (a) and composition of intracellular products of Synechocystis (conditions: Fe 2 O 3 : 20 µM; MgO: 200 µM and NADP: 150 µM; light: 100 µE/m 2 /s).

    Journal: bioRxiv

    Article Title: Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by Engineered Synechocystis sp. PCC 6803

    doi: 10.1101/529420

    Figure Lengend Snippet: Effect of incubation time on ethanol and biomass concentration (a) and composition of intracellular products of Synechocystis (conditions: Fe 2 O 3 : 20 µM; MgO: 200 µM and NADP: 150 µM; light: 100 µE/m 2 /s).

    Article Snippet: The filtered samples were used for total NADP (NADP and NADPH) and heated samples (60°C for 30 min) were used for NADPH analysis.

    Techniques: Incubation, Concentration Assay

    Effect of NADP concentration on biomass (a) and chlorophyll a (b) concentrations (conditions: cultivation time: 20 days; Fe 2 O 3 : 20 µM and MgO:200 µM).

    Journal: bioRxiv

    Article Title: Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by Engineered Synechocystis sp. PCC 6803

    doi: 10.1101/529420

    Figure Lengend Snippet: Effect of NADP concentration on biomass (a) and chlorophyll a (b) concentrations (conditions: cultivation time: 20 days; Fe 2 O 3 : 20 µM and MgO:200 µM).

    Article Snippet: The filtered samples were used for total NADP (NADP and NADPH) and heated samples (60°C for 30 min) were used for NADPH analysis.

    Techniques: Concentration Assay

    6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances the resistance of tumor cells to ionizing radiation (IR). a , b U87/EGFRvIII cells stably expressing luciferase were depleted of endogenous 6PGD and reconstituted with the expression of r6PGD WT or Y481F. These cells were treated with or without X-ray radiation (10 Gy) and cultured for 8 h. Reactive oxygen species (ROS) levels were measured using ROS assay kit ( a ). NADPH/NADP + ratio was determined using NADP + /NADPH quantitation kit ( b ). Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Journal: Nature Communications

    Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

    doi: 10.1038/s41467-019-08921-8

    Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances the resistance of tumor cells to ionizing radiation (IR). a , b U87/EGFRvIII cells stably expressing luciferase were depleted of endogenous 6PGD and reconstituted with the expression of r6PGD WT or Y481F. These cells were treated with or without X-ray radiation (10 Gy) and cultured for 8 h. Reactive oxygen species (ROS) levels were measured using ROS assay kit ( a ). NADPH/NADP + ratio was determined using NADP + /NADPH quantitation kit ( b ). Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Article Snippet: NADPH/NADP+ ratio NADPH/NADP+ ratios were determined by measuring NADPH/NADP+ concentrations according to the protocol of NADP/NADPH assay (Sigma).

    Techniques: Stable Transfection, Expressing, Luciferase, Cell Culture, ROS Assay, Quantitation Assay, Two Tailed Test

    6-Phosphogluconate dehydrogenase (6PGD) pY481 is required for EGF-enhanced 6PGD activity. a U87/epidermal growth factor receptor (EGFR) cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD were treated with or without EGF (100 ng ml -1 ) for 30 min. pTyr phospho-tyrosine, pSer phospho-serine, pThr phospho-threonine. b Immunoprecipitated Flag-6PGD from U87/EGFR cells treated with or without EGF (100 ng ml -1 ) for 30 min, was subjected to mass spectrometry analyses. Mass spectrometry analysis of a tryptic fragment at m/z 999.20270 ( z = + 4), matched to the charged peptide 1-DYFGAHTYELLAKPGQFIHTNWTGHGGTVSSSS(pY)NA-36. The probability of pY481 was 99.99%. c U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml -1 ) for 30 min. d U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with EGF (100 ng ml -1 ) for 30 min. e Sequence alignment of phosphorylated peptides among indicated species. f Representative image of structure of human 6PGD bound to NADP + (PDB ID:2JKV) was shown. Dimeric 6PGD was shown as ribbons and NADP + was shown as balls and sticks. 6PGD Y481 was shown as sticks. Cyan ribbon or gray ribbon represented two monomers in dimeric 6PGD, respectively. g , h Flag-6PGD WT or Y481F were immunoprecipitated from U87/EGFR cells with or without EGF (100 ng ml -1 ) treatment for 30 min. The enzymatic activity of immunoprecipitated Flag-6PGD proteins was examined ( g ). Statistical analyses of 6PGD activities ( h ). The activity of 6PGD was normalized against protein amounts. 6PGD WT activity without EGF treatment was normalized to 1.0. Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Journal: Nature Communications

    Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

    doi: 10.1038/s41467-019-08921-8

    Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase (6PGD) pY481 is required for EGF-enhanced 6PGD activity. a U87/epidermal growth factor receptor (EGFR) cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD were treated with or without EGF (100 ng ml -1 ) for 30 min. pTyr phospho-tyrosine, pSer phospho-serine, pThr phospho-threonine. b Immunoprecipitated Flag-6PGD from U87/EGFR cells treated with or without EGF (100 ng ml -1 ) for 30 min, was subjected to mass spectrometry analyses. Mass spectrometry analysis of a tryptic fragment at m/z 999.20270 ( z = + 4), matched to the charged peptide 1-DYFGAHTYELLAKPGQFIHTNWTGHGGTVSSSS(pY)NA-36. The probability of pY481 was 99.99%. c U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml -1 ) for 30 min. d U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with EGF (100 ng ml -1 ) for 30 min. e Sequence alignment of phosphorylated peptides among indicated species. f Representative image of structure of human 6PGD bound to NADP + (PDB ID:2JKV) was shown. Dimeric 6PGD was shown as ribbons and NADP + was shown as balls and sticks. 6PGD Y481 was shown as sticks. Cyan ribbon or gray ribbon represented two monomers in dimeric 6PGD, respectively. g , h Flag-6PGD WT or Y481F were immunoprecipitated from U87/EGFR cells with or without EGF (100 ng ml -1 ) treatment for 30 min. The enzymatic activity of immunoprecipitated Flag-6PGD proteins was examined ( g ). Statistical analyses of 6PGD activities ( h ). The activity of 6PGD was normalized against protein amounts. 6PGD WT activity without EGF treatment was normalized to 1.0. Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Article Snippet: NADPH/NADP+ ratio NADPH/NADP+ ratios were determined by measuring NADPH/NADP+ concentrations according to the protocol of NADP/NADPH assay (Sigma).

    Techniques: Activity Assay, Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry, Sequencing, Two Tailed Test

    6-Phosphogluconate dehydrogenase (6PGD) Y481 phosphorylation promotes DNA synthesis and glioma progression. a U87/EGFRvIII cells stably expressing luciferase were depleted of endogenous 6PGD and reconstituted with the expression of r6PGD WT or Y481F. The expression of 6PGD was examined using immunoblotting assays. b – e NADP + /NADPH ratio ( b ), relative reactive oxygen species (ROS) levels ( c ), cellular ribulose-5-phosphate (Ru-5-P) level ( d ), and BrdU incorporation ratio ( e ) were measured in these cells generated in a . f The flux through the pentose phosphate pathway (PPP) was determined using d -glucose-1,2- 13 C 2 in these cells generated in a . g Cellular ATP levels were determined in these cells generated in a . h , i Cell proliferation ( h ) and colony formation ( i ) were determined in these cells generated in a . j , k These cells generated in a (2 × 10 5 per mouse) were intracranially injected into randomized athymic nude mice (five mice per group). Bioluminescence imaging of tumor growth were carried out. Real time images were presented ( j , left panel) and the intensities of luciferase were quantified ( j , right panel). After 11 days, tumor growth was examined. Hematoxylin and eosin (H E)-stained coronal brain sections show representative tumor xenografts. Scale bar, 100 μm ( k , left panel). Representative images of tumor boundaries were presented with ×200 magnification. Tumor volumes were measured using length ( a ) and width ( b ) and calculated using the equation: V = ab 2 /2 ( k , right panel). Data represent the mean ± SD of luciferase intensity of five mice per group. Student’s t- test (unpaired, two tailed), ** p

    Journal: Nature Communications

    Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

    doi: 10.1038/s41467-019-08921-8

    Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase (6PGD) Y481 phosphorylation promotes DNA synthesis and glioma progression. a U87/EGFRvIII cells stably expressing luciferase were depleted of endogenous 6PGD and reconstituted with the expression of r6PGD WT or Y481F. The expression of 6PGD was examined using immunoblotting assays. b – e NADP + /NADPH ratio ( b ), relative reactive oxygen species (ROS) levels ( c ), cellular ribulose-5-phosphate (Ru-5-P) level ( d ), and BrdU incorporation ratio ( e ) were measured in these cells generated in a . f The flux through the pentose phosphate pathway (PPP) was determined using d -glucose-1,2- 13 C 2 in these cells generated in a . g Cellular ATP levels were determined in these cells generated in a . h , i Cell proliferation ( h ) and colony formation ( i ) were determined in these cells generated in a . j , k These cells generated in a (2 × 10 5 per mouse) were intracranially injected into randomized athymic nude mice (five mice per group). Bioluminescence imaging of tumor growth were carried out. Real time images were presented ( j , left panel) and the intensities of luciferase were quantified ( j , right panel). After 11 days, tumor growth was examined. Hematoxylin and eosin (H E)-stained coronal brain sections show representative tumor xenografts. Scale bar, 100 μm ( k , left panel). Representative images of tumor boundaries were presented with ×200 magnification. Tumor volumes were measured using length ( a ) and width ( b ) and calculated using the equation: V = ab 2 /2 ( k , right panel). Data represent the mean ± SD of luciferase intensity of five mice per group. Student’s t- test (unpaired, two tailed), ** p

    Article Snippet: NADPH/NADP+ ratio NADPH/NADP+ ratios were determined by measuring NADPH/NADP+ concentrations according to the protocol of NADP/NADPH assay (Sigma).

    Techniques: DNA Synthesis, Stable Transfection, Expressing, Luciferase, BrdU Incorporation Assay, Generated, Injection, Mouse Assay, Imaging, Staining, Two Tailed Test

    6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances NADP + binding affinity of 6PGD. a U87/epidermal growth factor receptor (EGFR) cells stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml -1 ) for 30 min. Cell lysates were incubated with Cibacron blue beads mimicking NADP + for a pulldown assay. b In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without active recombinant Fyn. ITC assays were performed with pulldown 6PGD variants (0.05 mM) and NADP + (1 mM) (top panel). His peptides were used as negative control. Statistical analyses of K d values of 6PGD variants for NADP + were presented (bottom panel). c , d In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without recombinant active Fyn. K m ( c ) and k cat ( d ) of 6PGD variants were determined. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. b – d Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Journal: Nature Communications

    Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

    doi: 10.1038/s41467-019-08921-8

    Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances NADP + binding affinity of 6PGD. a U87/epidermal growth factor receptor (EGFR) cells stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml -1 ) for 30 min. Cell lysates were incubated with Cibacron blue beads mimicking NADP + for a pulldown assay. b In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without active recombinant Fyn. ITC assays were performed with pulldown 6PGD variants (0.05 mM) and NADP + (1 mM) (top panel). His peptides were used as negative control. Statistical analyses of K d values of 6PGD variants for NADP + were presented (bottom panel). c , d In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without recombinant active Fyn. K m ( c ) and k cat ( d ) of 6PGD variants were determined. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. b – d Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p

    Article Snippet: NADPH/NADP+ ratio NADPH/NADP+ ratios were determined by measuring NADPH/NADP+ concentrations according to the protocol of NADP/NADPH assay (Sigma).

    Techniques: Binding Assay, Stable Transfection, Expressing, Incubation, In Vitro, Purification, Recombinant, Negative Control, Immunoprecipitation, Two Tailed Test

    miR-206 and miR-613 have rewired cellular metabolism by targeting 6PGD. (A,B) C13 ∗ (A) and A549DDP (B) cell lines were transfected with Mimic NC, miR-206 and miR-613, respectively. The effects of miRNAs on NADPH/NADP + ratio, lactate production and intracellular ATP levels were determined. (C,D) OV2008 (C) and A549 (D) cell lines were transfected with Inhibitor NC and miR-206 inhibitor and miR-613 inhibitor, respectively. The effects of miRNA inhibitors on NADPH/NADP + ratio, lactate production and intracellular ATP levels were determined. Error bars represent mean values ± SD from three replicates of each sample ( ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Inhibition of 6-phosphogluconate Dehydrogenase Reverses Cisplatin Resistance in Ovarian and Lung Cancer

    doi: 10.3389/fphar.2017.00421

    Figure Lengend Snippet: miR-206 and miR-613 have rewired cellular metabolism by targeting 6PGD. (A,B) C13 ∗ (A) and A549DDP (B) cell lines were transfected with Mimic NC, miR-206 and miR-613, respectively. The effects of miRNAs on NADPH/NADP + ratio, lactate production and intracellular ATP levels were determined. (C,D) OV2008 (C) and A549 (D) cell lines were transfected with Inhibitor NC and miR-206 inhibitor and miR-613 inhibitor, respectively. The effects of miRNA inhibitors on NADPH/NADP + ratio, lactate production and intracellular ATP levels were determined. Error bars represent mean values ± SD from three replicates of each sample ( ∗ P

    Article Snippet: NADPH/NADP+ Ratio Assay NADPH/NADP+ ratio was measured by a Colorimetric Assay Kit (Sigma–Aldrich) as described previously ( ).

    Techniques: Transfection

    6-phosphogluconate dehydrogenase offers superiority for cisplatin resistance. Proposed working model. (A) In cisplatin-resistant cancer cells, miRNA-206 and miRNA-613 are commonly downregulated in cisplatin-resistant cells, leading to reduced expression levels of 6PGD. And reprogramming cell metabolism, including NADPH/NADP+ ratio, lactate production and intracellular ATP levels, to fulfill cancer cells resistant to cisplatin treatment. (B) Attenuation of 6PGD by miRNAs or small molecule inhibitor Physcion results in decreased 6PGD expression or enzyme activity, leading to cancer cell sensitive to cisplatin treatment.

    Journal: Frontiers in Pharmacology

    Article Title: Inhibition of 6-phosphogluconate Dehydrogenase Reverses Cisplatin Resistance in Ovarian and Lung Cancer

    doi: 10.3389/fphar.2017.00421

    Figure Lengend Snippet: 6-phosphogluconate dehydrogenase offers superiority for cisplatin resistance. Proposed working model. (A) In cisplatin-resistant cancer cells, miRNA-206 and miRNA-613 are commonly downregulated in cisplatin-resistant cells, leading to reduced expression levels of 6PGD. And reprogramming cell metabolism, including NADPH/NADP+ ratio, lactate production and intracellular ATP levels, to fulfill cancer cells resistant to cisplatin treatment. (B) Attenuation of 6PGD by miRNAs or small molecule inhibitor Physcion results in decreased 6PGD expression or enzyme activity, leading to cancer cell sensitive to cisplatin treatment.

    Article Snippet: NADPH/NADP+ Ratio Assay NADPH/NADP+ ratio was measured by a Colorimetric Assay Kit (Sigma–Aldrich) as described previously ( ).

    Techniques: Expressing, Activity Assay

    Cisplatin-resistant cells have rewired cellular metabolism. (A) Sensitivity of OV2008, C13 ∗ , A549, and A549DDP cells upon 72 h cisplatin exposure were determined by cell counting. (B–E) OV2008, C13 ∗ , A549, and A549DDP cells were tested for oxidative PPP flux (B) , NADPH/NADP + ratio (C) , lactate production (D) , and intracellular ATP levels (E) . Error bars represent mean values ± SD from three replicates of each sample ( ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Inhibition of 6-phosphogluconate Dehydrogenase Reverses Cisplatin Resistance in Ovarian and Lung Cancer

    doi: 10.3389/fphar.2017.00421

    Figure Lengend Snippet: Cisplatin-resistant cells have rewired cellular metabolism. (A) Sensitivity of OV2008, C13 ∗ , A549, and A549DDP cells upon 72 h cisplatin exposure were determined by cell counting. (B–E) OV2008, C13 ∗ , A549, and A549DDP cells were tested for oxidative PPP flux (B) , NADPH/NADP + ratio (C) , lactate production (D) , and intracellular ATP levels (E) . Error bars represent mean values ± SD from three replicates of each sample ( ∗ P

    Article Snippet: NADPH/NADP+ Ratio Assay NADPH/NADP+ ratio was measured by a Colorimetric Assay Kit (Sigma–Aldrich) as described previously ( ).

    Techniques: Cell Counting

    L-arginine availability regulates redox balance in L . donovani promastigotes. (A-E) Leishmania parasites were grown in L-arginine depleted (AD- Ld ), L-arginine depleted but supplemented with ornithine (AD- Ld /Orn+), putrescine (AD- Ld /Put+), NAC (AD- Ld /NAC+) and L-arginine supplemented (AS- Ld ) RPMI media for 0–120 hr. Intracellular ROS production was measured using the fluorescent dye 2, 7-dicholorodihydroflourescein diacetate (H 2 DCFDA) and NAC was used as a ROS scavenger (A). The rate of lipid peroxidation was measured by spectrofluorometry and expressed as fluorescence units at 430 nm (B). The total intracellular reduced thiol levels were measured spectrophotometrically using DTNB (C). SOD activity was measured by using SOD assay kit as described in “Materials and methods (D). The relative NADP+/NADPH ratio was measured spectrophotometrically based on the measurement of the absorbance of the reduced coenzyme at 450 nm (E). The data represents mean±SD of triplicate determinations and are representative of three independent experiments.*, P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    doi: 10.1371/journal.pntd.0004373

    Figure Lengend Snippet: L-arginine availability regulates redox balance in L . donovani promastigotes. (A-E) Leishmania parasites were grown in L-arginine depleted (AD- Ld ), L-arginine depleted but supplemented with ornithine (AD- Ld /Orn+), putrescine (AD- Ld /Put+), NAC (AD- Ld /NAC+) and L-arginine supplemented (AS- Ld ) RPMI media for 0–120 hr. Intracellular ROS production was measured using the fluorescent dye 2, 7-dicholorodihydroflourescein diacetate (H 2 DCFDA) and NAC was used as a ROS scavenger (A). The rate of lipid peroxidation was measured by spectrofluorometry and expressed as fluorescence units at 430 nm (B). The total intracellular reduced thiol levels were measured spectrophotometrically using DTNB (C). SOD activity was measured by using SOD assay kit as described in “Materials and methods (D). The relative NADP+/NADPH ratio was measured spectrophotometrically based on the measurement of the absorbance of the reduced coenzyme at 450 nm (E). The data represents mean±SD of triplicate determinations and are representative of three independent experiments.*, P

    Article Snippet: Measurement of NADP+/NADPH ratio NADP+/NADPH ratio for the AD-Ld , AD-Ld /Orn+, AD-Ld /Put+, AD-Ld /NAC+ and AS-Ld parasites were assayed spectrophotometrically using NADP/NADPH Quantification Kit (Sigma) as per manufacturer’s instruction as described [ ].

    Techniques: Fluorescence, Activity Assay

    Schematic representation demonstrating the role of L-arginine in the modulation of cell proliferation or apoptosis-like cell death in Leishmania parasite. The model illustrates that the fate of Leishmania parasites depend on the availability of extracellular L-arginine. If L-arginine is available, it is taken up from the external medium by cationic L-arginine transporters (y+) and metabolized by arginase (AS) and other polyamine biosynthetic and thiol metabolic pathway enzymes such as γ-glutamylcysteine synthetase (γ-GCS), ornithine decarboxylase (ODC), spermidine synthase (SpdS), trypanothione synthetase (TryS), trypanothione reductase (TR), tryparedoxin (TXN) to produce L-ornithine (Orn), putrescine (Put), spermidine (Spd) and thiols such as trypanothione (TryP) which promotes parasite proliferation and cell growth. Whereas, if extracellular L-arginine is unavailable or deficient, cellular redox balance is altered characterized by increased NADP+/NADPH ratio, decreased antioxidant levels such as SOD activity and thiol content that led to increased ROS production. L-arginine unavailability also triggered the phenomenons like phosphatidyl serine externalization, increased lipid peroxidation and release of intracellular calcium. Calcium influx in turn causes mitochondrial membrane depolarization leading to release of cytochrome-c from mitochondrion and intracellular ROS generation that ultimately damages DNA and promotes an apoptosis-like cell death.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    doi: 10.1371/journal.pntd.0004373

    Figure Lengend Snippet: Schematic representation demonstrating the role of L-arginine in the modulation of cell proliferation or apoptosis-like cell death in Leishmania parasite. The model illustrates that the fate of Leishmania parasites depend on the availability of extracellular L-arginine. If L-arginine is available, it is taken up from the external medium by cationic L-arginine transporters (y+) and metabolized by arginase (AS) and other polyamine biosynthetic and thiol metabolic pathway enzymes such as γ-glutamylcysteine synthetase (γ-GCS), ornithine decarboxylase (ODC), spermidine synthase (SpdS), trypanothione synthetase (TryS), trypanothione reductase (TR), tryparedoxin (TXN) to produce L-ornithine (Orn), putrescine (Put), spermidine (Spd) and thiols such as trypanothione (TryP) which promotes parasite proliferation and cell growth. Whereas, if extracellular L-arginine is unavailable or deficient, cellular redox balance is altered characterized by increased NADP+/NADPH ratio, decreased antioxidant levels such as SOD activity and thiol content that led to increased ROS production. L-arginine unavailability also triggered the phenomenons like phosphatidyl serine externalization, increased lipid peroxidation and release of intracellular calcium. Calcium influx in turn causes mitochondrial membrane depolarization leading to release of cytochrome-c from mitochondrion and intracellular ROS generation that ultimately damages DNA and promotes an apoptosis-like cell death.

    Article Snippet: Measurement of NADP+/NADPH ratio NADP+/NADPH ratio for the AD-Ld , AD-Ld /Orn+, AD-Ld /Put+, AD-Ld /NAC+ and AS-Ld parasites were assayed spectrophotometrically using NADP/NADPH Quantification Kit (Sigma) as per manufacturer’s instruction as described [ ].

    Techniques: Activity Assay

    Intracellular polyP, ATP, NADP, and NADPH in the Δ phoU2 mutant. Bacteria were grown to exponential phase in TSB medium. PolyP was assessed by measuring the fluorescence emission of the DAPI-polyP complex at 550 nm. (A) The fluorescence (in arbitrary units [AU]) of the DAPI-polyP complex was measured at 550 nm to evaluate the amount of intracellular polyP. (B) The amount of ATP was determined by measuring the fluorescence emission of the ATP complex at 587 nm. Bacteria were grown to exponential phase in TSB medium (OD 600 = 0.5). (C and D) The amounts of total NADP and NADPH were measured using an NADP/NADPH quantification kit (Sigma). The amounts of total NADP and NADPH were determined by measuring the absorbance of the NADPH complex at 450 nm. Data (means ± SDs) are from three independent experiments. **, P

    Journal: Journal of Bacteriology

    Article Title: PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis

    doi: 10.1128/JB.00219-17

    Figure Lengend Snippet: Intracellular polyP, ATP, NADP, and NADPH in the Δ phoU2 mutant. Bacteria were grown to exponential phase in TSB medium. PolyP was assessed by measuring the fluorescence emission of the DAPI-polyP complex at 550 nm. (A) The fluorescence (in arbitrary units [AU]) of the DAPI-polyP complex was measured at 550 nm to evaluate the amount of intracellular polyP. (B) The amount of ATP was determined by measuring the fluorescence emission of the ATP complex at 587 nm. Bacteria were grown to exponential phase in TSB medium (OD 600 = 0.5). (C and D) The amounts of total NADP and NADPH were measured using an NADP/NADPH quantification kit (Sigma). The amounts of total NADP and NADPH were determined by measuring the absorbance of the NADPH complex at 450 nm. Data (means ± SDs) are from three independent experiments. **, P

    Article Snippet: Because most of the NADP was generated via PPP and intracellular NADPH, as a reduced form of NADP, plays a very important role in protecting against the toxicity of reactive oxygen species (ROS), intracellular NADPH levels in the ΔphoU2 mutant, the ΔphoU1 mutant, and the parent strain were determined using an NADP/NADPH quantification kit (Sigma).

    Techniques: Mutagenesis, Fluorescence

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M109.083428

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

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

    Techniques: Staining, Isolation, Immunohistochemistry

    DHRS2 decreased ROS in vitro . ( a ) NADP/NADPH ratio decreased in DHRS2-V1 and DHRS2-V2-transfected KYSE510 cells compared with vector control cells; NADP/NADPH ratio increased in DHRS2 knock-down KYSE180 and HKESC1 cells (* P

    Journal: Oncogene

    Article Title: DHRS2 inhibits cell growth and motility in esophageal squamous cell carcinoma

    doi: 10.1038/onc.2017.383

    Figure Lengend Snippet: DHRS2 decreased ROS in vitro . ( a ) NADP/NADPH ratio decreased in DHRS2-V1 and DHRS2-V2-transfected KYSE510 cells compared with vector control cells; NADP/NADPH ratio increased in DHRS2 knock-down KYSE180 and HKESC1 cells (* P

    Article Snippet: NADP/NADPH quantification NADP/NADPH quantification was detected in DHRS2-V1 and DHRS2-V2-overexpressed and knock-down cells as the manufacturer’s protocol (Sigma-Aldrich).

    Techniques: In Vitro, Transfection, Plasmid Preparation

    Localized chaperone deficiency does not activate any of the key cellular stress responses. Transcript levels of (a) cytosolic, (b) ER and (c) mitochondrial target genes in the chaperone deficient strains, compared to the WT. UBC6 was used as a control. The measurement was performed in biological and technical triplicate. (d) NADP/NADPH ratio is decreased in the chaperone deficient mutants. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p

    Journal: Scientific Reports

    Article Title: Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

    doi: 10.1038/srep28751

    Figure Lengend Snippet: Localized chaperone deficiency does not activate any of the key cellular stress responses. Transcript levels of (a) cytosolic, (b) ER and (c) mitochondrial target genes in the chaperone deficient strains, compared to the WT. UBC6 was used as a control. The measurement was performed in biological and technical triplicate. (d) NADP/NADPH ratio is decreased in the chaperone deficient mutants. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p

    Article Snippet: NADP/NADPH ratio was measured using NADP/NADPH quantitation kit (Sigma-Aldrich) according to the manufacturers instructions.

    Techniques:

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.48

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.48

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

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

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

    mTOR inhibition reduces NOX expression and antioxidants block oxLDL priming. Cells were treated with 100 nM Torin1 or vehicle an hour before treatment with 20 μg/ml oxLDL or vehicle. On day 3 cells were harvested and mRNA expression of NOX2 (A) and NOX4 (B) was analyzed using real-time qPCR. (C) Cells were treated as described above. Concentrations of NADP and NADPH were measured using a colorimetric assay kit on day 6. Ratio of NADP/NADPH is shown. (D,E) Monocytes were pre-incubated for 1 h with 0.5 μM Diphenyleneiodonium (DPI), 25 μM VAS2870, 40 μM Mito-TEMPO or vehicle before treating with oxLDL or vehicle for 24 h. On day 6 cells were stimulated with Pam3cys and IL6 (D) and TNFα (E) were measured in the supernatant using ELISA. Graphs represent mean values ± SD of at least 6 individuals in at least 3 different experiments. * P

    Journal: Frontiers in Immunology

    Article Title: mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes

    doi: 10.3389/fimmu.2018.03155

    Figure Lengend Snippet: mTOR inhibition reduces NOX expression and antioxidants block oxLDL priming. Cells were treated with 100 nM Torin1 or vehicle an hour before treatment with 20 μg/ml oxLDL or vehicle. On day 3 cells were harvested and mRNA expression of NOX2 (A) and NOX4 (B) was analyzed using real-time qPCR. (C) Cells were treated as described above. Concentrations of NADP and NADPH were measured using a colorimetric assay kit on day 6. Ratio of NADP/NADPH is shown. (D,E) Monocytes were pre-incubated for 1 h with 0.5 μM Diphenyleneiodonium (DPI), 25 μM VAS2870, 40 μM Mito-TEMPO or vehicle before treating with oxLDL or vehicle for 24 h. On day 6 cells were stimulated with Pam3cys and IL6 (D) and TNFα (E) were measured in the supernatant using ELISA. Graphs represent mean values ± SD of at least 6 individuals in at least 3 different experiments. * P

    Article Snippet: NADP/NADPH Assay NADP/NADPH levels were measured using a colorimetric NADP/NADPH assay kit (Abcam, #ab65349) according to the manufacturer's instructions.

    Techniques: Inhibition, Expressing, Blocking Assay, Real-time Polymerase Chain Reaction, Colorimetric Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Proposed model of SM-FDG uptake and its relationship with NADP reduction to NADPH. The cartoon proposes the ER contribution to FDG kinetics. Pathways of glucose and FDG are depicted in black and red, respectively. G, gluconate; 6PG, 6-P-gluconate; F-2D-G, fluoro-deoxy-gluconate; F-2D-PG, fluoro-deoxy-6P-gluconate. Gluts are depicted as squares, G6P-transporter as a circle.

    Journal: Molecular Metabolism

    Article Title: FDG uptake tracks the oxidative damage in diabetic skeletal muscle: An experimental study

    doi: 10.1016/j.molmet.2019.11.007

    Figure Lengend Snippet: Proposed model of SM-FDG uptake and its relationship with NADP reduction to NADPH. The cartoon proposes the ER contribution to FDG kinetics. Pathways of glucose and FDG are depicted in black and red, respectively. G, gluconate; 6PG, 6-P-gluconate; F-2D-G, fluoro-deoxy-gluconate; F-2D-PG, fluoro-deoxy-6P-gluconate. Gluts are depicted as squares, G6P-transporter as a circle.

    Article Snippet: Glutathione reductase (GR) activity and the NADPH/NADP ratio in SM homogenates were evaluated spectrophotometrically, at 405 and 450 nm, respectively, using a GR assay kit (Abcam: ab83461) and NADP-NADPH assay kit (Abcam: ab65349), following the manufacturer's instructions.

    Techniques:

    Effect of diabetes and metformin on the enzymatic pathway. Catalytic activities of HK (A), PFK (B), G6PD (C), and H6PD (D) in control (solid columns) and STZ-DM groups (dashed columns) of untreated (green) or treated with low-dose (red) or high-dose (blue) MTF (right) SM homogenate. The NADPH/NADP ratio is represented in panel E. The correlation between H6PD activity and MRGlu (F). GR and G6Pase catalytic activities are expressed in panels G and H, respectively. * p

    Journal: Molecular Metabolism

    Article Title: FDG uptake tracks the oxidative damage in diabetic skeletal muscle: An experimental study

    doi: 10.1016/j.molmet.2019.11.007

    Figure Lengend Snippet: Effect of diabetes and metformin on the enzymatic pathway. Catalytic activities of HK (A), PFK (B), G6PD (C), and H6PD (D) in control (solid columns) and STZ-DM groups (dashed columns) of untreated (green) or treated with low-dose (red) or high-dose (blue) MTF (right) SM homogenate. The NADPH/NADP ratio is represented in panel E. The correlation between H6PD activity and MRGlu (F). GR and G6Pase catalytic activities are expressed in panels G and H, respectively. * p

    Article Snippet: Glutathione reductase (GR) activity and the NADPH/NADP ratio in SM homogenates were evaluated spectrophotometrically, at 405 and 450 nm, respectively, using a GR assay kit (Abcam: ab83461) and NADP-NADPH assay kit (Abcam: ab65349), following the manufacturer's instructions.

    Techniques: Activity Assay

    mTOR inhibition reduces NOX expression and antioxidants block oxLDL priming. Cells were treated with 100 nM Torin1 or vehicle an hour before treatment with 20 μg/ml oxLDL or vehicle. On day 3 cells were harvested and mRNA expression of NOX2 (A) and NOX4 (B) was analyzed using real-time qPCR. (C) Cells were treated as described above. Concentrations of NADP and NADPH were measured using a colorimetric assay kit on day 6. Ratio of NADP/NADPH is shown. (D,E) Monocytes were pre-incubated for 1 h with 0.5 μM Diphenyleneiodonium (DPI), 25 μM VAS2870, 40 μM Mito-TEMPO or vehicle before treating with oxLDL or vehicle for 24 h. On day 6 cells were stimulated with Pam3cys and IL6 (D) and TNFα (E) were measured in the supernatant using ELISA. Graphs represent mean values ± SD of at least 6 individuals in at least 3 different experiments. * P

    Journal: Frontiers in Immunology

    Article Title: mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes

    doi: 10.3389/fimmu.2018.03155

    Figure Lengend Snippet: mTOR inhibition reduces NOX expression and antioxidants block oxLDL priming. Cells were treated with 100 nM Torin1 or vehicle an hour before treatment with 20 μg/ml oxLDL or vehicle. On day 3 cells were harvested and mRNA expression of NOX2 (A) and NOX4 (B) was analyzed using real-time qPCR. (C) Cells were treated as described above. Concentrations of NADP and NADPH were measured using a colorimetric assay kit on day 6. Ratio of NADP/NADPH is shown. (D,E) Monocytes were pre-incubated for 1 h with 0.5 μM Diphenyleneiodonium (DPI), 25 μM VAS2870, 40 μM Mito-TEMPO or vehicle before treating with oxLDL or vehicle for 24 h. On day 6 cells were stimulated with Pam3cys and IL6 (D) and TNFα (E) were measured in the supernatant using ELISA. Graphs represent mean values ± SD of at least 6 individuals in at least 3 different experiments. * P

    Article Snippet: NADP/NADPH levels were measured using a colorimetric NADP/NADPH assay kit (Abcam, #ab65349) according to the manufacturer's instructions.

    Techniques: Inhibition, Expressing, Blocking Assay, Real-time Polymerase Chain Reaction, Colorimetric Assay, Incubation, Enzyme-linked Immunosorbent Assay

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

    Journal:

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

    doi: 10.1161/ATVBAHA.109.197822

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

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

    Techniques: Expressing, Inhibition, Activity Assay

    CO inhibits PDGF-stimulated NADPH oxidase activity

    Journal:

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

    doi: 10.1161/ATVBAHA.109.197822

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

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

    Techniques: Activity Assay