nadp nadph ratio  (Abcam)

 
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    NADPH
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    Structured Review

    Abcam nadp nadph ratio
    HuR regulates IDH1 expression in PDAC A, RNA sequencing was performed in two PDAC cell lines (HS-766T and MiaPaCa2) modulated by CRISPR/Cas9 against HuR. The heatmap displays transcripts encoding antioxidant enzymes (out of 40 directly involved in ROS detoxification), with significant changes in HuR(−/−) cells as compared with the HuR(+/+) control cells. HuR (ELAVL1, the CRISPR deletion target) is included in the table as reference. B, Schematic of the reversible IDH1 catalytic reaction. <t>NADPH</t> is produced by oxidative decarboxylation. C, HuR and IDH1 mRNA levels in MiaPaca2 CRISPR HuR(+/+) or HuR(−/−) cells; immunoblot analysis of IDH1 and HuR protein in the same cells. D, MiaPaCa2 cells were cotransfected with siRNAs and luciferase reporter constructs (luciferase control or fused with IDH1 3′ UTR). Cells were cultured as indicated for 24 hours. E, αKG levels in MiaPaCa2 cells cultured under the indicated conditions for 24 hours. Error bars, ± SEM of triplicate wells from a representative experiment. *, P

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    Images

    1) Product Images from "Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells"

    Article Title: Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-17-0015

    HuR regulates IDH1 expression in PDAC A, RNA sequencing was performed in two PDAC cell lines (HS-766T and MiaPaCa2) modulated by CRISPR/Cas9 against HuR. The heatmap displays transcripts encoding antioxidant enzymes (out of 40 directly involved in ROS detoxification), with significant changes in HuR(−/−) cells as compared with the HuR(+/+) control cells. HuR (ELAVL1, the CRISPR deletion target) is included in the table as reference. B, Schematic of the reversible IDH1 catalytic reaction. NADPH is produced by oxidative decarboxylation. C, HuR and IDH1 mRNA levels in MiaPaca2 CRISPR HuR(+/+) or HuR(−/−) cells; immunoblot analysis of IDH1 and HuR protein in the same cells. D, MiaPaCa2 cells were cotransfected with siRNAs and luciferase reporter constructs (luciferase control or fused with IDH1 3′ UTR). Cells were cultured as indicated for 24 hours. E, αKG levels in MiaPaCa2 cells cultured under the indicated conditions for 24 hours. Error bars, ± SEM of triplicate wells from a representative experiment. *, P
    Figure Legend Snippet: HuR regulates IDH1 expression in PDAC A, RNA sequencing was performed in two PDAC cell lines (HS-766T and MiaPaCa2) modulated by CRISPR/Cas9 against HuR. The heatmap displays transcripts encoding antioxidant enzymes (out of 40 directly involved in ROS detoxification), with significant changes in HuR(−/−) cells as compared with the HuR(+/+) control cells. HuR (ELAVL1, the CRISPR deletion target) is included in the table as reference. B, Schematic of the reversible IDH1 catalytic reaction. NADPH is produced by oxidative decarboxylation. C, HuR and IDH1 mRNA levels in MiaPaca2 CRISPR HuR(+/+) or HuR(−/−) cells; immunoblot analysis of IDH1 and HuR protein in the same cells. D, MiaPaCa2 cells were cotransfected with siRNAs and luciferase reporter constructs (luciferase control or fused with IDH1 3′ UTR). Cells were cultured as indicated for 24 hours. E, αKG levels in MiaPaCa2 cells cultured under the indicated conditions for 24 hours. Error bars, ± SEM of triplicate wells from a representative experiment. *, P

    Techniques Used: Expressing, RNA Sequencing Assay, CRISPR, Produced, Luciferase, Construct, Cell Culture

    HuR minimizes ROS levels, while enhancing mitochondrial function A, NADP + /NADPH ratio in MiaPaCa2 cells cultured under the indicated conditions for 24 hours. B, GSSG/GSH ratio in MiaPaCa2 cells under the same conditions. C, ROS levels, detected by DCF fluorescence in MiaPaCa2 cells cultured in 5 mmol/L glucose media for 24 hours, and gemcitabine (GEM; 1 µmol/L) as indicated. D, Mitochondrial-specific ROS were analyzed using MitoSOX in MiaPaCa2 cells cultured in 5 mmol/L glucose media for 24 hours, and gemcitabine (1 µmol/L) as indicated. E , Oxygen consumption rates (OCR) in MiaPaCa2 cells, cultured in 5 mmol/L glucose for 24 hours. F, Quantitative 13 C-isotope tracer labeling of TCA cycle intermediates (or surrogate metabolites, e.g., Asp) in MiaPaca2 cells that were modified by CRISPR/Cas9 against HuR(−/−), or isogenic controls (+/+). Cells were cultured in 5 mmol/L glucose for 24 hours in the presence of [1,2- 13 C 6 ]glucose. Each data point represents the mean ± SEM of three independent experiments. *, P
    Figure Legend Snippet: HuR minimizes ROS levels, while enhancing mitochondrial function A, NADP + /NADPH ratio in MiaPaCa2 cells cultured under the indicated conditions for 24 hours. B, GSSG/GSH ratio in MiaPaCa2 cells under the same conditions. C, ROS levels, detected by DCF fluorescence in MiaPaCa2 cells cultured in 5 mmol/L glucose media for 24 hours, and gemcitabine (GEM; 1 µmol/L) as indicated. D, Mitochondrial-specific ROS were analyzed using MitoSOX in MiaPaCa2 cells cultured in 5 mmol/L glucose media for 24 hours, and gemcitabine (1 µmol/L) as indicated. E , Oxygen consumption rates (OCR) in MiaPaCa2 cells, cultured in 5 mmol/L glucose for 24 hours. F, Quantitative 13 C-isotope tracer labeling of TCA cycle intermediates (or surrogate metabolites, e.g., Asp) in MiaPaca2 cells that were modified by CRISPR/Cas9 against HuR(−/−), or isogenic controls (+/+). Cells were cultured in 5 mmol/L glucose for 24 hours in the presence of [1,2- 13 C 6 ]glucose. Each data point represents the mean ± SEM of three independent experiments. *, P

    Techniques Used: Cell Culture, Fluorescence, Labeling, Modification, CRISPR

    2) Product Images from "TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells"

    Article Title: TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells

    Journal: Scientific Reports

    doi: 10.1038/srep35016

    TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P
    Figure Legend Snippet: TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Western Blot, Immunofluorescence, Confocal Microscopy

    3) Product Images from "mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03155

    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
    Figure Legend 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

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

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

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

    Journal: Reproductive Sciences

    doi: 10.1177/1933719114537712

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

    Techniques Used:

    5) Product Images from "Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells"

    Article Title: Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.204

    Identification of estradiol-induced metabolic signature of pentose phosphate pathway in Tsc2-deficient cells in vitro . ( a ) ELT3 (Tsc2-deficient rat uterus-derived) cells were treated with estradiol (10 nM) for 2 and 24 h. Cellular metabolites were profiled by mass spectrometry ( n =5) (Metabolon LC-MS/MS). ( b ) Box-plots of glucose, glucose-6-phosphate, fructose-6-phophate, ribose, ribose-5-phosphate, and ribulose-5-phosphate are shown. Data show the mean of five sets of independent samples. ( c ) Cellular NADPH levels were measured in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 2 and 24 h. Data were normalized to total protein level. Results are representative of three sets of independent samples per group from three experiments. ( d ) Cellular ROS levels were measured using DCFH-DA in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 24 h. Cells were incubated with hydrogen peroxide (H 2 O 2 ) (0.5 μ M) for 1 h before measurement. Data were normalized to total cell number. Results are representative of eight independent samples per group from three experiments. ( e ) Extracellular lactate levels were measured in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 24 h. FBS (10%) stimulation was included as a positive control. Lactate levels were normalized to total proteins. Results are representative of three sets of independent samples per group from three experiments. ( f ) Patient-derived TSC2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with hydrogen peroxide (0.5 μ M) for 0.5 h. Cell morphology was recorded using phase-contrast microscopy. ( g ) Patient-derived TSC2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with H 2 O 2 (0.5 μ M) for 0.5 h. Cell death was measured using the propidium iodide (PI) exclusion assay. Proportion of dead cells was normalized to the total number of variable cells. Results are representative of eight independent samples per group from three experiments. ( h ) Rat-derived Tsc2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with hydrogen peroxide (0.5 μ M) for 1 h. Cell morphology was recorded using phase-contrast microscopy. ( i ) Rat-derived Tsc2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with H 2 O 2 (0.5 μ M) for 1 h. Cell death was measured using the PI exclusion assay. Proportion of dead cells was normalized to the total number of variable cells. Results are representative of eight independent samples per group from three experiments. ( j ) Scheme of glucose metabolism in TSC2-deficient cells treated with estradiol. * P
    Figure Legend Snippet: Identification of estradiol-induced metabolic signature of pentose phosphate pathway in Tsc2-deficient cells in vitro . ( a ) ELT3 (Tsc2-deficient rat uterus-derived) cells were treated with estradiol (10 nM) for 2 and 24 h. Cellular metabolites were profiled by mass spectrometry ( n =5) (Metabolon LC-MS/MS). ( b ) Box-plots of glucose, glucose-6-phosphate, fructose-6-phophate, ribose, ribose-5-phosphate, and ribulose-5-phosphate are shown. Data show the mean of five sets of independent samples. ( c ) Cellular NADPH levels were measured in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 2 and 24 h. Data were normalized to total protein level. Results are representative of three sets of independent samples per group from three experiments. ( d ) Cellular ROS levels were measured using DCFH-DA in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 24 h. Cells were incubated with hydrogen peroxide (H 2 O 2 ) (0.5 μ M) for 1 h before measurement. Data were normalized to total cell number. Results are representative of eight independent samples per group from three experiments. ( e ) Extracellular lactate levels were measured in rat-derived Tsc2-deficient cells treated with control or estradiol (10 nM) for 24 h. FBS (10%) stimulation was included as a positive control. Lactate levels were normalized to total proteins. Results are representative of three sets of independent samples per group from three experiments. ( f ) Patient-derived TSC2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with hydrogen peroxide (0.5 μ M) for 0.5 h. Cell morphology was recorded using phase-contrast microscopy. ( g ) Patient-derived TSC2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with H 2 O 2 (0.5 μ M) for 0.5 h. Cell death was measured using the propidium iodide (PI) exclusion assay. Proportion of dead cells was normalized to the total number of variable cells. Results are representative of eight independent samples per group from three experiments. ( h ) Rat-derived Tsc2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with hydrogen peroxide (0.5 μ M) for 1 h. Cell morphology was recorded using phase-contrast microscopy. ( i ) Rat-derived Tsc2-deficient cells were treated with control or estradiol (10 nM) for 24 h, and then incubated with H 2 O 2 (0.5 μ M) for 1 h. Cell death was measured using the PI exclusion assay. Proportion of dead cells was normalized to the total number of variable cells. Results are representative of eight independent samples per group from three experiments. ( j ) Scheme of glucose metabolism in TSC2-deficient cells treated with estradiol. * P

    Techniques Used: In Vitro, Derivative Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, Positive Control, Microscopy, Exclusion Assay

    Depletion of G6PD attenuates estradiol-enhanced survival of Tsc2-deficient cells in vitro and in vivo . ( a ) Tsc2-deficient cells were transfected with control siRNA or G6PD siRNA for 48 h, and then treated with control or estradiol for 6 h. Levels of G6PD, phospho-S6 (Ser235/236), and β -actin were assessed by immunoblot. The graph indicates the ratio of G6PD to β -actin. Results are representative of three different experiments. ( b ) Tsc2-deficient cells were transfected with control siRNA or G6PD siRNA for 48 h, and then treated with control or estradiol for 24 h. Hydrogen peroxide (0.5 μ M) was added for 1 h before measurement. Cellular ROS level was measured with the DCFH-DA. Data were normalized to total cell number. Results are representative of eight sets of independent samples per group from three experiments. ( c ) Cell death was measured using propidium iodide (PI) exclusion assay. Data were normalized to total cell number. Results are representative of eight sets of independent samples per group from three experiments. ( d ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol for 24 h. G6PD activity was determined. ( e ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 2 and 24 h. Cellular NADPH level was measured. Data were normalized to total protein level. Results are representative of three sets of independent samples per group from three experiments. ( f ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 24 h. Cellular ROS level was measured with the DCFH-DA. Data were normalized to cell number. Results are representative of eight sets of independent samples per group from three experiments. ( g ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 24 h. Cell death was measured using the PI exclusion assay. Data were normalized to cell number. Results are representative of eight sets of independent samples per group from three experiments. ( h ) Female CB17 SCID mice were supplemented with estradiol for 1 week, and then treated with 6-AN or vehicle for 2 days. ELT3-luciferase cells were treated with 6-AN for 18 h, and then injected intravenously into mice ( n =5/group). Lung colonization was measured using bioluminescence at 0.5, 6, and 24 h after injection. Representative images are shown. Total photon flux/second present in the chest regions in estradiol ( n =5) and estradiol plus 6-AN-treated ( n =5) animals. ( i ) Scheme of estradiol-promoted pentose phosphate pathway addiction in TSC2-deficient cells. * P
    Figure Legend Snippet: Depletion of G6PD attenuates estradiol-enhanced survival of Tsc2-deficient cells in vitro and in vivo . ( a ) Tsc2-deficient cells were transfected with control siRNA or G6PD siRNA for 48 h, and then treated with control or estradiol for 6 h. Levels of G6PD, phospho-S6 (Ser235/236), and β -actin were assessed by immunoblot. The graph indicates the ratio of G6PD to β -actin. Results are representative of three different experiments. ( b ) Tsc2-deficient cells were transfected with control siRNA or G6PD siRNA for 48 h, and then treated with control or estradiol for 24 h. Hydrogen peroxide (0.5 μ M) was added for 1 h before measurement. Cellular ROS level was measured with the DCFH-DA. Data were normalized to total cell number. Results are representative of eight sets of independent samples per group from three experiments. ( c ) Cell death was measured using propidium iodide (PI) exclusion assay. Data were normalized to total cell number. Results are representative of eight sets of independent samples per group from three experiments. ( d ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol for 24 h. G6PD activity was determined. ( e ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 2 and 24 h. Cellular NADPH level was measured. Data were normalized to total protein level. Results are representative of three sets of independent samples per group from three experiments. ( f ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 24 h. Cellular ROS level was measured with the DCFH-DA. Data were normalized to cell number. Results are representative of eight sets of independent samples per group from three experiments. ( g ) Tsc2-deficient cells were treated with 6-AN (10 μ M) for 1 h, and then treated with control or estradiol (10 nM) for 24 h. Cell death was measured using the PI exclusion assay. Data were normalized to cell number. Results are representative of eight sets of independent samples per group from three experiments. ( h ) Female CB17 SCID mice were supplemented with estradiol for 1 week, and then treated with 6-AN or vehicle for 2 days. ELT3-luciferase cells were treated with 6-AN for 18 h, and then injected intravenously into mice ( n =5/group). Lung colonization was measured using bioluminescence at 0.5, 6, and 24 h after injection. Representative images are shown. Total photon flux/second present in the chest regions in estradiol ( n =5) and estradiol plus 6-AN-treated ( n =5) animals. ( i ) Scheme of estradiol-promoted pentose phosphate pathway addiction in TSC2-deficient cells. * P

    Techniques Used: In Vitro, In Vivo, Transfection, Exclusion Assay, Activity Assay, Mouse Assay, Luciferase, Injection

    6) Product Images from "Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis"

    Article Title: Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar4062

    Cardiac mitochondrial NADP+-dependent isocitrate dehydrogenase (mNADP-ICDH) activity (A), protein (B), and mRNA (C) . Total proteins and RNA were extracted from left ventricular (LV) tissues of control and adjuvant-induced arthritis (AIA) rats and processed to enzymatic assay (A) , Western blotting (B) or real-time PCR (C) , as described under Materials and Methods. (D) Cardiac 4-hydroxynonenal (HNE)/NADP-ICDH adducts were identified in total proteins by immunoprecipitation as described in Material and Methods. Values are the mean ± SEM of three experiments. ** P
    Figure Legend Snippet: Cardiac mitochondrial NADP+-dependent isocitrate dehydrogenase (mNADP-ICDH) activity (A), protein (B), and mRNA (C) . Total proteins and RNA were extracted from left ventricular (LV) tissues of control and adjuvant-induced arthritis (AIA) rats and processed to enzymatic assay (A) , Western blotting (B) or real-time PCR (C) , as described under Materials and Methods. (D) Cardiac 4-hydroxynonenal (HNE)/NADP-ICDH adducts were identified in total proteins by immunoprecipitation as described in Material and Methods. Values are the mean ± SEM of three experiments. ** P

    Techniques Used: Activity Assay, Enzymatic Assay, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation

    Determination of cardiac redox status . Glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) were determined in total proteins extracted in left ventricular (LV) tissues from control and adjuvant-induced arthritis (AIA) rats. Data are mean ± SEM of three experiments and expressed as GSSG/(GSSG+GSH) and NADP/(NADP+NADPH) ratio. Statistics: one-way ANOVA; * P
    Figure Legend Snippet: Determination of cardiac redox status . Glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) were determined in total proteins extracted in left ventricular (LV) tissues from control and adjuvant-induced arthritis (AIA) rats. Data are mean ± SEM of three experiments and expressed as GSSG/(GSSG+GSH) and NADP/(NADP+NADPH) ratio. Statistics: one-way ANOVA; * P

    Techniques Used:

    Modulation of mitochondrial NADP+-dependent isocitrate dehydrogenase (mNADP-ICDH), type I collagen (Col I) and connective tissue growth factor (CTGF) in isolated adult rat cardiomyocytes . Cells were treated with 10 μM 4-hydroxynonenal (HNE) for 48 hours in the presence or absence of 10 μM ramipril and then total proteins were preceded to the determination of (A) NADP-ICDH activity, (B) HNE/NADP-ICDH adducts, and (C) Col I and CTGF protein as described in Materials and Methods. Values are the mean ± SEM of three experiments. * P
    Figure Legend Snippet: Modulation of mitochondrial NADP+-dependent isocitrate dehydrogenase (mNADP-ICDH), type I collagen (Col I) and connective tissue growth factor (CTGF) in isolated adult rat cardiomyocytes . Cells were treated with 10 μM 4-hydroxynonenal (HNE) for 48 hours in the presence or absence of 10 μM ramipril and then total proteins were preceded to the determination of (A) NADP-ICDH activity, (B) HNE/NADP-ICDH adducts, and (C) Col I and CTGF protein as described in Materials and Methods. Values are the mean ± SEM of three experiments. * P

    Techniques Used: Isolation, Activity Assay

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

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

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00611

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

    Techniques Used: Labeling, Staining, Fluorescence, Infection

    8) Product Images from "DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function"

    Article Title: DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.31

    The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. ( a ) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS ( b – g ) and A549 ( h – m ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS ( b ) and A549 cells ( h ). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS ( c ) and A549 cells ( i ). Forty-eight hours after siRNA transfection, NAD + , NADH, NADP + and NADPH levels were analysed using colorimetric assays in U2OS ( d – g ) and A549 cells ( j – m ). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t -test (paired, one tailed) (* P
    Figure Legend Snippet: The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. ( a ) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS ( b – g ) and A549 ( h – m ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS ( b ) and A549 cells ( h ). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS ( c ) and A549 cells ( i ). Forty-eight hours after siRNA transfection, NAD + , NADH, NADP + and NADPH levels were analysed using colorimetric assays in U2OS ( d – g ) and A549 cells ( j – m ). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t -test (paired, one tailed) (* P

    Techniques Used: Transfection, Positive Control, One-tailed Test

    9) Product Images from "mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03155

    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
    Figure Legend 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

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

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

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

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00611

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

    Techniques Used: Labeling, Staining, Fluorescence, Infection

    11) Product Images from "mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03155

    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
    Figure Legend 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

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

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

    13) Product Images from "The mitochondrial carrier SLC25A10 regulates cancer cell growth"

    Article Title: The mitochondrial carrier SLC25A10 regulates cancer cell growth

    Journal: Oncotarget

    doi:

    Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p
    Figure Legend Snippet: Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p

    Techniques Used:

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

    15) Product Images from "Mutual Regulation of Epicardial Adipose Tissue and Myocardial Redox State by PPAR-γ/Adiponectin Signalling"

    Article Title: Mutual Regulation of Epicardial Adipose Tissue and Myocardial Redox State by PPAR-γ/Adiponectin Signalling

    Journal: Circulation Research

    doi: 10.1161/CIRCRESAHA.115.307856

    Circulating adiponectin (AdN) is positively related with myocardial redox state in patients with ischemic heart disease. In the Clinical Associations Studies, high circulating AdN levels were paradoxically related with high myocardial nicotinamide adenine dinucleotide phosphate (NADPH)–stimulated superoxide (O 2 − ) ( A ) and high plasma malonyldialdehyde (MDA; a marker of systemic oxidative stress; B ). There was no association of circulating adiponectin with plasma interleukin-6 (IL-6; C ) or high-sensitivity C-reactive protein (hsCRP; D ). High circulating adiponectin was also positively related with high plasma brain natriuretic peptide (BNP; E ). Values are expressed as median (25th–75th percentile).
    Figure Legend Snippet: Circulating adiponectin (AdN) is positively related with myocardial redox state in patients with ischemic heart disease. In the Clinical Associations Studies, high circulating AdN levels were paradoxically related with high myocardial nicotinamide adenine dinucleotide phosphate (NADPH)–stimulated superoxide (O 2 − ) ( A ) and high plasma malonyldialdehyde (MDA; a marker of systemic oxidative stress; B ). There was no association of circulating adiponectin with plasma interleukin-6 (IL-6; C ) or high-sensitivity C-reactive protein (hsCRP; D ). High circulating adiponectin was also positively related with high plasma brain natriuretic peptide (BNP; E ). Values are expressed as median (25th–75th percentile).

    Techniques Used: Multiple Displacement Amplification, Marker

    Myocardial oxidation product 4-hydroxynonenal (4HNE) as a mediator of the inside-to-outside signal from the human myocardium to epicardial adipose tissue (EpAT). In human myocardium, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidases activity (upper tertile of NADPH-stimulated O 2 − in the Clinical Associations Studies) were associated with significantly greater 4HNE ( A ) and malonyldialdehyde (MDA; B ) production. Incubation of EpAT and thoracic (ThAT) adipose tissue with MDA (1 mmol/L) for 16 h had no significant impact on ADIPOQ gene expression ( C ). On the contrary, incubation of EpAT with 4HNE (30 µmol/L) for 16 h induced a striking increase in ADIPOQ ( D ), PPAR-γ ( E ) and CD36 ( F ). The effects of 4HNE on ADIPOQ and CD36 gene expression were reversed by the inhibitor of PPAR-γ activity, T0070907 (10 μmol/L; D and F ). Importantly, 4HNE had no significant impact on the expression of ADIPOQ ( G ), PPAR-γ ( H ), or CD36 ( I ) genes in ThAT. Values are represented as fold change compared to control group (mean±SEM). A and B , n=4 per group; ( C ) n=5 per group; ( D – I ), n=5 to 8 per group, * P
    Figure Legend Snippet: Myocardial oxidation product 4-hydroxynonenal (4HNE) as a mediator of the inside-to-outside signal from the human myocardium to epicardial adipose tissue (EpAT). In human myocardium, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidases activity (upper tertile of NADPH-stimulated O 2 − in the Clinical Associations Studies) were associated with significantly greater 4HNE ( A ) and malonyldialdehyde (MDA; B ) production. Incubation of EpAT and thoracic (ThAT) adipose tissue with MDA (1 mmol/L) for 16 h had no significant impact on ADIPOQ gene expression ( C ). On the contrary, incubation of EpAT with 4HNE (30 µmol/L) for 16 h induced a striking increase in ADIPOQ ( D ), PPAR-γ ( E ) and CD36 ( F ). The effects of 4HNE on ADIPOQ and CD36 gene expression were reversed by the inhibitor of PPAR-γ activity, T0070907 (10 μmol/L; D and F ). Importantly, 4HNE had no significant impact on the expression of ADIPOQ ( G ), PPAR-γ ( H ), or CD36 ( I ) genes in ThAT. Values are represented as fold change compared to control group (mean±SEM). A and B , n=4 per group; ( C ) n=5 per group; ( D – I ), n=5 to 8 per group, * P

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Incubation, Expressing

    Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in H9C2 cardiomyocytes triggers peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling in rat pericardial fat: identifying a novel inside-to-outside signal. To examine whether under conditions of increased endogenous oxidative stress cardiac myocytes release a transferable factor able to affect the activation of PPAR-γ/adiponectin signaling in rat epicardial adipose tissue (EpAT), we exposed H9c2 cells (differentiated to cardiac myocytes) to NADPH 100 μmol/L for 2 h, whereas rat EpAT was conditioned ex vivo ( A ). After 2 h, the rat EpAT was transferred into the H9c2 wells and cocultured for an additional 16 h ( A ). At the end of the incubation period, gene expression was studied in the rat EpAT. Addition of NADPH to intact H9c2 cells grown on coverslips led to a striking increase of NADPH oxidase–derived superoxide (O 2 − ) that was partly inhibitable by either Vas2870 (a pan-Nox inhibitor) or gp91-dstat (a specific inhibitor of NOX2; B ), as demonstrated by real-time monitoring using lucigenin-enhanced chemiluminescence. Coincubation of rat EpAT with H9c2 cardiac myocytes stimulated with NADPH resulted in an upregulation of ADIPOQ ( C ), PPAR-γ ( D ), and CD36 ( E ) in EpAT at 16 h. All these effects were prevented by polyethylene glycol (PEG)-SOD (300 U/mL) or vas2870 (10 nmol/L). The presence of unstimulated H9C2 cells or NADPH alone had no effect on the expression of ADIPOQ, PPAR-γ , or CD36 genes in the rat EpAT ( C – E ). Concentration of gp91-dstat was 50 μmol/L. Values are presented as mean±SEM. B , n=7; ( C – E ), n=7; * P
    Figure Legend Snippet: Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in H9C2 cardiomyocytes triggers peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling in rat pericardial fat: identifying a novel inside-to-outside signal. To examine whether under conditions of increased endogenous oxidative stress cardiac myocytes release a transferable factor able to affect the activation of PPAR-γ/adiponectin signaling in rat epicardial adipose tissue (EpAT), we exposed H9c2 cells (differentiated to cardiac myocytes) to NADPH 100 μmol/L for 2 h, whereas rat EpAT was conditioned ex vivo ( A ). After 2 h, the rat EpAT was transferred into the H9c2 wells and cocultured for an additional 16 h ( A ). At the end of the incubation period, gene expression was studied in the rat EpAT. Addition of NADPH to intact H9c2 cells grown on coverslips led to a striking increase of NADPH oxidase–derived superoxide (O 2 − ) that was partly inhibitable by either Vas2870 (a pan-Nox inhibitor) or gp91-dstat (a specific inhibitor of NOX2; B ), as demonstrated by real-time monitoring using lucigenin-enhanced chemiluminescence. Coincubation of rat EpAT with H9c2 cardiac myocytes stimulated with NADPH resulted in an upregulation of ADIPOQ ( C ), PPAR-γ ( D ), and CD36 ( E ) in EpAT at 16 h. All these effects were prevented by polyethylene glycol (PEG)-SOD (300 U/mL) or vas2870 (10 nmol/L). The presence of unstimulated H9C2 cells or NADPH alone had no effect on the expression of ADIPOQ, PPAR-γ , or CD36 genes in the rat EpAT ( C – E ). Concentration of gp91-dstat was 50 μmol/L. Values are presented as mean±SEM. B , n=7; ( C – E ), n=7; * P

    Techniques Used: Activation Assay, Ex Vivo, Incubation, Expressing, Derivative Assay, Concentration Assay

    Effects of recombinant adiponectin (AdN) on myocardial redox state in humans. Ex vivo incubation of human myocardium with AdN (10 μg/mL) for 2 h resulted in increased phosphorylation of AMP-kinase (AMPK)-α at Thr172 (p-AMPK; A ) leading to AMPK activation as assessed by the phosphorylation status of its downstream target acetyl-CoA carboxylase (ACC) at Ser79 (p-ACC; B ), an effect reversed by compound C (CC; 10 μmol/L; A and B ). AdN reduced superoxide (O 2 − ) production in human myocardium ( C ) and specifically nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as assessed by measuring the Vas2870-inhibitable (40 μmol/L) O 2 − ( D ); both effects were reversed by CC ( C and D ). These effects of AdN on myocardial O 2 − were also confirmed by dihydroethidium (DHE) staining; AdN reduced both total and Vas2870-inhibitable DHE fluorescence, and these effects were reversed by CC ( E – G ). Importantly, AdN prevented Rac1 activation (assessed by measuring the ratio of GTP-Rac1:total Rac1 [t-Rac1; H ]) and reduced the membrane-bound fraction of Rac1 (m-Rac1; I ); both effects were reversed by CC. Similarly, AdN prevented p47 phox phosphorylation at its activatory site Ser359 (p-p47 phox , J ) and reduced the membrane-bound fraction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47 phox (m-p47 phox ; K ) in an AMPK-dependent manner (as both effects were reversed by CC; J and K ). A and B , AdN: n=7 to 13 per group; CC: 4 to 7 per group; ( C , D , H , J , K ) AdN, n=7 to 12; CC group, n=4 to 6; ( E – I ) n=3 to 4 per group. Values are expressed as fold change vs control group and shown as mean±SEM; * P
    Figure Legend Snippet: Effects of recombinant adiponectin (AdN) on myocardial redox state in humans. Ex vivo incubation of human myocardium with AdN (10 μg/mL) for 2 h resulted in increased phosphorylation of AMP-kinase (AMPK)-α at Thr172 (p-AMPK; A ) leading to AMPK activation as assessed by the phosphorylation status of its downstream target acetyl-CoA carboxylase (ACC) at Ser79 (p-ACC; B ), an effect reversed by compound C (CC; 10 μmol/L; A and B ). AdN reduced superoxide (O 2 − ) production in human myocardium ( C ) and specifically nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as assessed by measuring the Vas2870-inhibitable (40 μmol/L) O 2 − ( D ); both effects were reversed by CC ( C and D ). These effects of AdN on myocardial O 2 − were also confirmed by dihydroethidium (DHE) staining; AdN reduced both total and Vas2870-inhibitable DHE fluorescence, and these effects were reversed by CC ( E – G ). Importantly, AdN prevented Rac1 activation (assessed by measuring the ratio of GTP-Rac1:total Rac1 [t-Rac1; H ]) and reduced the membrane-bound fraction of Rac1 (m-Rac1; I ); both effects were reversed by CC. Similarly, AdN prevented p47 phox phosphorylation at its activatory site Ser359 (p-p47 phox , J ) and reduced the membrane-bound fraction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47 phox (m-p47 phox ; K ) in an AMPK-dependent manner (as both effects were reversed by CC; J and K ). A and B , AdN: n=7 to 13 per group; CC: 4 to 7 per group; ( C , D , H , J , K ) AdN, n=7 to 12; CC group, n=4 to 6; ( E – I ) n=3 to 4 per group. Values are expressed as fold change vs control group and shown as mean±SEM; * P

    Techniques Used: Recombinant, Ex Vivo, Incubation, Activation Assay, Activity Assay, Staining, Fluorescence

    Genetically conferred increases in adiponectin (AdN) bioavailability are causally associated with lower nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the human myocardium. The total number of rs17366568G alleles (polymorphism in ADIPOQ gene) and rs266717T alleles (polymorphism in ADIPOQ gene promoter) had an additive effect on circulating AdN levels ( A ) and was associated with reduced NADPH–stimulated superoxide (O 2 − ) in human myocardium ( B ). The number of rs17366568G/rs266717T alleles was positively associated with higher ADIPOQ gene expression in thoracic adipose tissue (ThAT, C ), but not associated in epicardial adipose tissue (EpAT; D ). Patients with higher ADIPOQ ( E ) or peroxisome proliferator-activated receptor (PPAR)-γ ( F ) gene expression in EpAT also had higher NADPH-stimulated O 2 − production in their myocardium. Higher myocardial NADPH-stimulated O 2 − was not associated with endogenous ADIPOQ gene expression in the heart ( G ), but was associated with higher gene expression of adiponectin receptor-1 (AdipoR1), but not of AdipoR2 or T-cadherin (CDH13) in human myocardial tissue ( H ). Values are expressed as median (25th–75th percentile). RLU indicates relative light units.
    Figure Legend Snippet: Genetically conferred increases in adiponectin (AdN) bioavailability are causally associated with lower nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the human myocardium. The total number of rs17366568G alleles (polymorphism in ADIPOQ gene) and rs266717T alleles (polymorphism in ADIPOQ gene promoter) had an additive effect on circulating AdN levels ( A ) and was associated with reduced NADPH–stimulated superoxide (O 2 − ) in human myocardium ( B ). The number of rs17366568G/rs266717T alleles was positively associated with higher ADIPOQ gene expression in thoracic adipose tissue (ThAT, C ), but not associated in epicardial adipose tissue (EpAT; D ). Patients with higher ADIPOQ ( E ) or peroxisome proliferator-activated receptor (PPAR)-γ ( F ) gene expression in EpAT also had higher NADPH-stimulated O 2 − production in their myocardium. Higher myocardial NADPH-stimulated O 2 − was not associated with endogenous ADIPOQ gene expression in the heart ( G ), but was associated with higher gene expression of adiponectin receptor-1 (AdipoR1), but not of AdipoR2 or T-cadherin (CDH13) in human myocardial tissue ( H ). Values are expressed as median (25th–75th percentile). RLU indicates relative light units.

    Techniques Used: Activity Assay, Expressing

    Testing the inside-to-outside paracrine effects of the heart on adipose tissue using a pig model of rapid atrial pacing (RAP). In a pig model of RAP, myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidases activity was significantly higher in the paced animals than in sham, as shown by both the NADPH-stimulated and Vas2870-inhibitable superoxide (O 2 − ) signal ( A and B ). RAP also increased formation of 4-hydroxynonenal (4HNE; but no malonyldialdehyde [MDA]) protein adducts ( C and D ) and upregulated ADIPOQ gene expression in epicardial AT (EpAT) but not in remote AT depots, eg, ScAT ( E ). There was no difference in endogenous ADIPOQ gene expression levels in the heart ( F ) or myocardial expression of adiponectin receptors ( G ) between sham-operated and RAP animals; all panels, n=5 per group, * P
    Figure Legend Snippet: Testing the inside-to-outside paracrine effects of the heart on adipose tissue using a pig model of rapid atrial pacing (RAP). In a pig model of RAP, myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidases activity was significantly higher in the paced animals than in sham, as shown by both the NADPH-stimulated and Vas2870-inhibitable superoxide (O 2 − ) signal ( A and B ). RAP also increased formation of 4-hydroxynonenal (4HNE; but no malonyldialdehyde [MDA]) protein adducts ( C and D ) and upregulated ADIPOQ gene expression in epicardial AT (EpAT) but not in remote AT depots, eg, ScAT ( E ). There was no difference in endogenous ADIPOQ gene expression levels in the heart ( F ) or myocardial expression of adiponectin receptors ( G ) between sham-operated and RAP animals; all panels, n=5 per group, * P

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Expressing

    Testing the inside-to-outside paracrine effects of the heart on pericardial adipose tissue using a cardiomyocyte-specific Nox2-tg mouse model. In the cardiac myocyte–specific NOX2-transgenic mouse, myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidases were activated, as assessed by both the NADPH-stimulated ( A ) and Vas2870-inhibitable superoxide (O 2 − ) signal ( B ), and by increased formation of 4-hydroxynonenal (4HNE) protein adducts when compared with wild-type (wt) animals ( C ). There was no difference in the myocardial protein levels of malonyldialdehyde (MDA) adducts ( D ). Increased myocardial oxidative stress and 4HNE adducts formation in mNOX2 -tg mice led to increased ADIPOQ gene expression in the fat attached to the heart (pericardial adipose tissue [PerAT]), but not in remote AT depots, eg, subcutaneous AT (ScAT; E ). mNOX2 -tg mice also had increased endogenous levels of ADIPOQ gene expression in myocardial tissue ( F ), but there was no difference in the myocardial gene expression levels of any of adiponectin receptors, T-cadherin (CDH13), AdipoR1 and AdipoR2 ( G ); ( A – E ), n=5 to 6 per group; ( F and G ) n=9 to 10 per group, * P
    Figure Legend Snippet: Testing the inside-to-outside paracrine effects of the heart on pericardial adipose tissue using a cardiomyocyte-specific Nox2-tg mouse model. In the cardiac myocyte–specific NOX2-transgenic mouse, myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidases were activated, as assessed by both the NADPH-stimulated ( A ) and Vas2870-inhibitable superoxide (O 2 − ) signal ( B ), and by increased formation of 4-hydroxynonenal (4HNE) protein adducts when compared with wild-type (wt) animals ( C ). There was no difference in the myocardial protein levels of malonyldialdehyde (MDA) adducts ( D ). Increased myocardial oxidative stress and 4HNE adducts formation in mNOX2 -tg mice led to increased ADIPOQ gene expression in the fat attached to the heart (pericardial adipose tissue [PerAT]), but not in remote AT depots, eg, subcutaneous AT (ScAT; E ). mNOX2 -tg mice also had increased endogenous levels of ADIPOQ gene expression in myocardial tissue ( F ), but there was no difference in the myocardial gene expression levels of any of adiponectin receptors, T-cadherin (CDH13), AdipoR1 and AdipoR2 ( G ); ( A – E ), n=5 to 6 per group; ( F and G ) n=9 to 10 per group, * P

    Techniques Used: Transgenic Assay, Multiple Displacement Amplification, Mouse Assay, Expressing

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

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

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

    Journal: Reproductive Sciences

    doi: 10.1177/1933719114537712

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

    Techniques Used:

    19) Product Images from "Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis"

    Article Title: Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis

    Journal: EJNMMI Research

    doi: 10.1186/s13550-020-00666-6

    Antioxidant response and oxidative stress in SOD1 G93A mutation. a , b NADPH levels and total NADP+NADPH content in control (green column) and SOD1 G93A (red column) quadricep homogenates represented as percent of control. c GR activity and d MDA content in control (green column) and SOD1 G93A (red column) quadriceps. e Mean fluorescence index (MFI) of H 2 DCFDA in control and SOD1 G93A quadriceps muscles. f Immunofluorescence representative images of MitoTracker® (as specific mitochondria staining—red fluorescence), H 2 DCFDA (as specific ROS staining—green fluorescence), and colocalization staining signals, in control and SOD1 G93A quadriceps. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation
    Figure Legend Snippet: Antioxidant response and oxidative stress in SOD1 G93A mutation. a , b NADPH levels and total NADP+NADPH content in control (green column) and SOD1 G93A (red column) quadricep homogenates represented as percent of control. c GR activity and d MDA content in control (green column) and SOD1 G93A (red column) quadriceps. e Mean fluorescence index (MFI) of H 2 DCFDA in control and SOD1 G93A quadriceps muscles. f Immunofluorescence representative images of MitoTracker® (as specific mitochondria staining—red fluorescence), H 2 DCFDA (as specific ROS staining—green fluorescence), and colocalization staining signals, in control and SOD1 G93A quadriceps. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation

    Techniques Used: Mutagenesis, Activity Assay, Multiple Displacement Amplification, Fluorescence, Immunofluorescence, Staining

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

    21) Product Images from "DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function"

    Article Title: DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.31

    The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. ( a ) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS ( b – g ) and A549 ( h – m ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS ( b ) and A549 cells ( h ). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS ( c ) and A549 cells ( i ). Forty-eight hours after siRNA transfection, NAD + , NADH, NADP + and NADPH levels were analysed using colorimetric assays in U2OS ( d – g ) and A549 cells ( j – m ). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t -test (paired, one tailed) (* P
    Figure Legend Snippet: The absence of DAPK2 leads to reduced oxidative phosphorylation in U2OS and A549 cells. ( a ) Simplified cartoon depicting cellular metabolism pathways with glycolysis as the first step of glucose breakdown, and oxidative phosphorylation and anaerobic respiration as subsequent steps. To quantify cellular metabolic processes, U2OS ( b – g ) and A549 ( h – m ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection cells were analysed using a Seahorse Analyser. ECAR, an indirect measurement of lactic acid production, is depicted as fold change of mpH/min and normalised to siNS control in U2OS ( b ) and A549 cells ( h ). OCR, which can be used to determine mitochondrial respiration, is shown as fold change of pmol/min and normalised to siNS control in U2OS ( c ) and A549 cells ( i ). Forty-eight hours after siRNA transfection, NAD + , NADH, NADP + and NADPH levels were analysed using colorimetric assays in U2OS ( d – g ) and A549 cells ( j – m ). Treatment with MPP+ (1 mM, 24 h) served as a positive control. Data represent mean±S.E.M. of three independent experiments, statistical analyses were done using Student's t -test (paired, one tailed) (* P

    Techniques Used: Transfection, Positive Control, One-tailed Test

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

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800547R

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

    Techniques Used: Incubation

    23) Product Images from "Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages"

    Article Title: Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages

    Journal: Chemico-biological interactions

    doi: 10.1016/j.cbi.2017.01.012

    AR regulates NADPH levels in BMMs Levels of cellular NADPH in WT-BMMs than AR-null BMMs. Experiment were repeated a minimum of 3 times, and the data represent mean ± SEM, *P
    Figure Legend Snippet: AR regulates NADPH levels in BMMs Levels of cellular NADPH in WT-BMMs than AR-null BMMs. Experiment were repeated a minimum of 3 times, and the data represent mean ± SEM, *P

    Techniques Used:

    24) Product Images from "The mitochondrial carrier SLC25A10 regulates cancer cell growth"

    Article Title: The mitochondrial carrier SLC25A10 regulates cancer cell growth

    Journal: Oncotarget

    doi:

    Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p
    Figure Legend Snippet: Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p

    Techniques Used:

    25) Product Images from "Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule"

    Article Title: Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI129903

    MIOX overexpression promotes lysosomal permeability and decreases GSH concentration, GPX4 activity, and NADPH levels in cisplatin-treated HK-2 cells. Lysosomal permeability was investigated by AO staining. AO-associated green fluorescence in the cytoplasm increased, while red fluorescence in the lysosome decreased, suggesting increased lysosomal permeability of HK-2 cells following cisplatin treatment ( B vs. A ). The changes in fluorescence were accentuated in cisplatin-treated MIOX-overexpressing cells but attenuated in cisplatin-treated cells transfected with MIOX siRNA ( C – F ). The expression of GPX4, a key enzyme for ferroptosis inhibition, decreased after cisplatin treatment ( M ). A substantial decline in GPX4 activity was also observed in both HK-2 cells and MIOX-overexpressing cells, and this decrease was negated by the concomitant transfection with MIOX siRNA ( N ) ( n = 6; * P
    Figure Legend Snippet: MIOX overexpression promotes lysosomal permeability and decreases GSH concentration, GPX4 activity, and NADPH levels in cisplatin-treated HK-2 cells. Lysosomal permeability was investigated by AO staining. AO-associated green fluorescence in the cytoplasm increased, while red fluorescence in the lysosome decreased, suggesting increased lysosomal permeability of HK-2 cells following cisplatin treatment ( B vs. A ). The changes in fluorescence were accentuated in cisplatin-treated MIOX-overexpressing cells but attenuated in cisplatin-treated cells transfected with MIOX siRNA ( C – F ). The expression of GPX4, a key enzyme for ferroptosis inhibition, decreased after cisplatin treatment ( M ). A substantial decline in GPX4 activity was also observed in both HK-2 cells and MIOX-overexpressing cells, and this decrease was negated by the concomitant transfection with MIOX siRNA ( N ) ( n = 6; * P

    Techniques Used: Over Expression, Permeability, Concentration Assay, Activity Assay, Staining, Fluorescence, Transfection, Expressing, Inhibition

    Overexpression of MIOX exacerbates, while its gene disruption alleviates, renal tubular injury, lipid hydroperoxidation, and decline in GPX4 activity and NADPH levels in cisplatin-induced AKI. The expression profile of MIOX in WT, MIOX-Tg, and MIOX-KO mice was demonstrated by immunoblotting studies ( M ). Cisplatin treatment led to severe renal tubular injury in WT mice, which was accentuated in MIOX-Tg mice but attenuated in MIOX-KO mice ( A – F ). Similarly, NGAL mRNA levels increased in cisplatin-treated WT mice and MIOX-Tg mice, and a minimal increase was observed in cisplatin-treated MIOX-KO mice ( P ) ( n = 4; * P
    Figure Legend Snippet: Overexpression of MIOX exacerbates, while its gene disruption alleviates, renal tubular injury, lipid hydroperoxidation, and decline in GPX4 activity and NADPH levels in cisplatin-induced AKI. The expression profile of MIOX in WT, MIOX-Tg, and MIOX-KO mice was demonstrated by immunoblotting studies ( M ). Cisplatin treatment led to severe renal tubular injury in WT mice, which was accentuated in MIOX-Tg mice but attenuated in MIOX-KO mice ( A – F ). Similarly, NGAL mRNA levels increased in cisplatin-treated WT mice and MIOX-Tg mice, and a minimal increase was observed in cisplatin-treated MIOX-KO mice ( P ) ( n = 4; * P

    Techniques Used: Over Expression, Activity Assay, Expressing, Mouse Assay

    26) Product Images from "Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells"

    Article Title: Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00291

    NOX2 is a major contributor to ATP-induced ROS in GECs. (A) Measure of NADP/NADPH ratio in ATP (3 mM) treated primary GEC lysates at 2, 4, and 24 h. Increased NADP/NADPH ratio corresponds to more consumed NADPH. N = 3, * p
    Figure Legend Snippet: NOX2 is a major contributor to ATP-induced ROS in GECs. (A) Measure of NADP/NADPH ratio in ATP (3 mM) treated primary GEC lysates at 2, 4, and 24 h. Increased NADP/NADPH ratio corresponds to more consumed NADPH. N = 3, * p

    Techniques Used:

    P. gingivalis modulates NADPH consumption in GECs. Measure of NADP/NADPH ratio in P. gingivalis (MOI100) infected GECs with or without ATP (3 mM) pre-treatment. N = 3, * p
    Figure Legend Snippet: P. gingivalis modulates NADPH consumption in GECs. Measure of NADP/NADPH ratio in P. gingivalis (MOI100) infected GECs with or without ATP (3 mM) pre-treatment. N = 3, * p

    Techniques Used: Infection

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

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

    Journal: Reproductive Sciences

    doi: 10.1177/1933719114537712

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

    Techniques Used:

    28) Product Images from "The mitochondrial carrier SLC25A10 regulates cancer cell growth"

    Article Title: The mitochondrial carrier SLC25A10 regulates cancer cell growth

    Journal: Oncotarget

    doi:

    Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p
    Figure Legend Snippet: Total intracellular NADPt, NADPH levels and NADPH/NADPt ratios in SLC25A10 knockdown A549 cells at different growth situations (A) NADPt levels in dividing, confluent and confluent cells with glutamine starvation; (B) NADPH levels in dividing, confluent and confluent cells with glutamine starvation and (C) NADPH/NADPt ratios in dividing, confluent and confluent cells with glutamine starvation. Data represented as relative change of NADPt, NADPH and NADPH/NADPt in siRNA-SLC cells compared to siRNA-CON cells. All experiments are repeated 3 times independently, all data are presented as mean ± SD. * represents p

    Techniques Used:

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

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

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00611

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

    Techniques Used: Labeling, Staining, Fluorescence, Infection

    30) Product Images from "Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells"

    Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13028-1

    FASN deficit impairs the upregulation of glycolysis and the anaplerotic shift of Krebs cycle. a The lack of FASN led to an accumulation of NADPH, acetyl-, and malonyl-CoA. The increase in acetyl-CoA can allosterically inhibit the activity of pyruvate dehydrogenase. This can block the entry of pyruvate into the Krebs cycle (associated with a decrease in the NAD-NADH cycling, respiration, and glycolytic rates) and the derivation of carbon skeletons to biosynthetic routes. In addition, a decrease in Krebs cycle activity could be coupled to a limited ability to satisfy high energetic demands. Pyruvate accumulation could also decrease glycolysis, which in addition to limiting ATP production would impair the ability to derive carbon skeletons to the nucleotide synthesis pathways. All these processes are upregulated in transformed compared with normal cells and are a hallmark of cancer 33 . b Total abundance of acetyl-CoA and NADPH determined by LC/MS across the four genotypes ( n = 4). c Representative extracellular acidification rate (ECAR) measurements. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the indicated time point for each experiment. d Mitochondrial respiration reflected by oxygen consumption rate levels (OCR) was detected in the four genotypes. In c , d presented data are mean values ± SEM. e Mitotracker dye (green) was used to reveal the localization of mitochondria (left), and the positively charged dye TMRE (red) (middle) was used to compare mitochondrial transmembrane polarization. Representative fluorescence images of TMRE and Mitotracker were taken by confocal microscopy. Merged images are shown. Scale bars, 10 μM. Quantitative analysis of relative fluorescence intensity is shown in the right-hand side (FASN lox/lox -Empty, n = 388 cells; FASN ∆/∆ -Empty, n = 458 cells; FASN lox/lox -PyMT, n = 294 cells; FASN ∆/∆ -PyMT, n = 382 cells). FCCP was added to FASN lox/lox -PyMT as a negative control ( n = 90 cells). f Representative flow cytometry analysis (left) showing constitutive mitochondrial superoxide levels for each genotype ( n = 4). The quantitation of MitoSOX red fluorescence intensity is shown in the right. In b , e , and f represented data are the mean values ± SD. * P
    Figure Legend Snippet: FASN deficit impairs the upregulation of glycolysis and the anaplerotic shift of Krebs cycle. a The lack of FASN led to an accumulation of NADPH, acetyl-, and malonyl-CoA. The increase in acetyl-CoA can allosterically inhibit the activity of pyruvate dehydrogenase. This can block the entry of pyruvate into the Krebs cycle (associated with a decrease in the NAD-NADH cycling, respiration, and glycolytic rates) and the derivation of carbon skeletons to biosynthetic routes. In addition, a decrease in Krebs cycle activity could be coupled to a limited ability to satisfy high energetic demands. Pyruvate accumulation could also decrease glycolysis, which in addition to limiting ATP production would impair the ability to derive carbon skeletons to the nucleotide synthesis pathways. All these processes are upregulated in transformed compared with normal cells and are a hallmark of cancer 33 . b Total abundance of acetyl-CoA and NADPH determined by LC/MS across the four genotypes ( n = 4). c Representative extracellular acidification rate (ECAR) measurements. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the indicated time point for each experiment. d Mitochondrial respiration reflected by oxygen consumption rate levels (OCR) was detected in the four genotypes. In c , d presented data are mean values ± SEM. e Mitotracker dye (green) was used to reveal the localization of mitochondria (left), and the positively charged dye TMRE (red) (middle) was used to compare mitochondrial transmembrane polarization. Representative fluorescence images of TMRE and Mitotracker were taken by confocal microscopy. Merged images are shown. Scale bars, 10 μM. Quantitative analysis of relative fluorescence intensity is shown in the right-hand side (FASN lox/lox -Empty, n = 388 cells; FASN ∆/∆ -Empty, n = 458 cells; FASN lox/lox -PyMT, n = 294 cells; FASN ∆/∆ -PyMT, n = 382 cells). FCCP was added to FASN lox/lox -PyMT as a negative control ( n = 90 cells). f Representative flow cytometry analysis (left) showing constitutive mitochondrial superoxide levels for each genotype ( n = 4). The quantitation of MitoSOX red fluorescence intensity is shown in the right. In b , e , and f represented data are the mean values ± SD. * P

    Techniques Used: Activity Assay, Blocking Assay, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Fluorescence, Confocal Microscopy, Negative Control, Flow Cytometry, Cytometry, Quantitation Assay

    31) Product Images from "mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03155

    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
    Figure Legend 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

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

    32) Product Images from "Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis"

    Article Title: Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis

    Journal: EJNMMI Research

    doi: 10.1186/s13550-020-00666-6

    Antioxidant response and oxidative stress in SOD1 G93A mutation. a , b NADPH levels and total NADP+NADPH content in control (green column) and SOD1 G93A (red column) quadricep homogenates represented as percent of control. c GR activity and d MDA content in control (green column) and SOD1 G93A (red column) quadriceps. e Mean fluorescence index (MFI) of H 2 DCFDA in control and SOD1 G93A quadriceps muscles. f Immunofluorescence representative images of MitoTracker® (as specific mitochondria staining—red fluorescence), H 2 DCFDA (as specific ROS staining—green fluorescence), and colocalization staining signals, in control and SOD1 G93A quadriceps. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation
    Figure Legend Snippet: Antioxidant response and oxidative stress in SOD1 G93A mutation. a , b NADPH levels and total NADP+NADPH content in control (green column) and SOD1 G93A (red column) quadricep homogenates represented as percent of control. c GR activity and d MDA content in control (green column) and SOD1 G93A (red column) quadriceps. e Mean fluorescence index (MFI) of H 2 DCFDA in control and SOD1 G93A quadriceps muscles. f Immunofluorescence representative images of MitoTracker® (as specific mitochondria staining—red fluorescence), H 2 DCFDA (as specific ROS staining—green fluorescence), and colocalization staining signals, in control and SOD1 G93A quadriceps. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation

    Techniques Used: Mutagenesis, Activity Assay, Multiple Displacement Amplification, Fluorescence, Immunofluorescence, Staining

    33) Product Images from "TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells"

    Article Title: TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells

    Journal: Scientific Reports

    doi: 10.1038/srep35016

    TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P
    Figure Legend Snippet: TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Western Blot, Immunofluorescence, Confocal Microscopy

    34) Product Images from "Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule"

    Article Title: Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI129903

    MIOX overexpression promotes lysosomal permeability and decreases GSH concentration, GPX4 activity, and NADPH levels in cisplatin-treated HK-2 cells. Lysosomal permeability was investigated by AO staining. AO-associated green fluorescence in the cytoplasm increased, while red fluorescence in the lysosome decreased, suggesting increased lysosomal permeability of HK-2 cells following cisplatin treatment ( B vs. A ). The changes in fluorescence were accentuated in cisplatin-treated MIOX-overexpressing cells but attenuated in cisplatin-treated cells transfected with MIOX siRNA ( C – F ). The expression of GPX4, a key enzyme for ferroptosis inhibition, decreased after cisplatin treatment ( M ). A substantial decline in GPX4 activity was also observed in both HK-2 cells and MIOX-overexpressing cells, and this decrease was negated by the concomitant transfection with MIOX siRNA ( N ) ( n = 6; * P
    Figure Legend Snippet: MIOX overexpression promotes lysosomal permeability and decreases GSH concentration, GPX4 activity, and NADPH levels in cisplatin-treated HK-2 cells. Lysosomal permeability was investigated by AO staining. AO-associated green fluorescence in the cytoplasm increased, while red fluorescence in the lysosome decreased, suggesting increased lysosomal permeability of HK-2 cells following cisplatin treatment ( B vs. A ). The changes in fluorescence were accentuated in cisplatin-treated MIOX-overexpressing cells but attenuated in cisplatin-treated cells transfected with MIOX siRNA ( C – F ). The expression of GPX4, a key enzyme for ferroptosis inhibition, decreased after cisplatin treatment ( M ). A substantial decline in GPX4 activity was also observed in both HK-2 cells and MIOX-overexpressing cells, and this decrease was negated by the concomitant transfection with MIOX siRNA ( N ) ( n = 6; * P

    Techniques Used: Over Expression, Permeability, Concentration Assay, Activity Assay, Staining, Fluorescence, Transfection, Expressing, Inhibition

    Overexpression of MIOX exacerbates, while its gene disruption alleviates, renal tubular injury, lipid hydroperoxidation, and decline in GPX4 activity and NADPH levels in cisplatin-induced AKI. The expression profile of MIOX in WT, MIOX-Tg, and MIOX-KO mice was demonstrated by immunoblotting studies ( M ). Cisplatin treatment led to severe renal tubular injury in WT mice, which was accentuated in MIOX-Tg mice but attenuated in MIOX-KO mice ( A – F ). Similarly, NGAL mRNA levels increased in cisplatin-treated WT mice and MIOX-Tg mice, and a minimal increase was observed in cisplatin-treated MIOX-KO mice ( P ) ( n = 4; * P
    Figure Legend Snippet: Overexpression of MIOX exacerbates, while its gene disruption alleviates, renal tubular injury, lipid hydroperoxidation, and decline in GPX4 activity and NADPH levels in cisplatin-induced AKI. The expression profile of MIOX in WT, MIOX-Tg, and MIOX-KO mice was demonstrated by immunoblotting studies ( M ). Cisplatin treatment led to severe renal tubular injury in WT mice, which was accentuated in MIOX-Tg mice but attenuated in MIOX-KO mice ( A – F ). Similarly, NGAL mRNA levels increased in cisplatin-treated WT mice and MIOX-Tg mice, and a minimal increase was observed in cisplatin-treated MIOX-KO mice ( P ) ( n = 4; * P

    Techniques Used: Over Expression, Activity Assay, Expressing, Mouse Assay

    35) Product Images from "Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages"

    Article Title: Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages

    Journal: Chemico-biological interactions

    doi: 10.1016/j.cbi.2017.01.012

    AR regulates NADPH levels in BMMs Levels of cellular NADPH in WT-BMMs than AR-null BMMs. Experiment were repeated a minimum of 3 times, and the data represent mean ± SEM, *P
    Figure Legend Snippet: AR regulates NADPH levels in BMMs Levels of cellular NADPH in WT-BMMs than AR-null BMMs. Experiment were repeated a minimum of 3 times, and the data represent mean ± SEM, *P

    Techniques Used:

    36) Product Images from "Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells"

    Article Title: Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00291

    NOX2 is a major contributor to ATP-induced ROS in GECs. (A) Measure of NADP/NADPH ratio in ATP (3 mM) treated primary GEC lysates at 2, 4, and 24 h. Increased NADP/NADPH ratio corresponds to more consumed NADPH. N = 3, * p
    Figure Legend Snippet: NOX2 is a major contributor to ATP-induced ROS in GECs. (A) Measure of NADP/NADPH ratio in ATP (3 mM) treated primary GEC lysates at 2, 4, and 24 h. Increased NADP/NADPH ratio corresponds to more consumed NADPH. N = 3, * p

    Techniques Used:

    P. gingivalis modulates NADPH consumption in GECs. Measure of NADP/NADPH ratio in P. gingivalis (MOI100) infected GECs with or without ATP (3 mM) pre-treatment. N = 3, * p
    Figure Legend Snippet: P. gingivalis modulates NADPH consumption in GECs. Measure of NADP/NADPH ratio in P. gingivalis (MOI100) infected GECs with or without ATP (3 mM) pre-treatment. N = 3, * p

    Techniques Used: Infection

    Related Articles

    Generated:

    Article Title: CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION
    Article Snippet: .. Because of an excess of imputed values for NAD+ and NADP+ in the Metabolon generated dataset, we measured these metabolites via commercial enzymatic assay kits (Abcam) according to the manufacturer’s instructions. .. Antibodies for western blot analysis against AMPKα1/2, phospho-AMPKα1/2 (Thr 172), rS6, and phospho-rS6 (Ser 235/236) were obtained from Cell Signaling Technologies.

    other:

    Article Title: Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis
    Article Snippet: Analysis of NADP/NADPH Ratio in Zebrafish Embryos The NADP/NADPH ratio assay was performed on zebrafish tissue extracts using the NADP/NADPH assay kit (Abcam).

    Article Title: mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes
    Article Snippet: NADP/NADPH levels were measured using a colorimetric NADP/NADPH assay kit (Abcam, #ab65349) according to the manufacturer's instructions.

    Article Title: Metyrapone Alleviates Deleterious Effects of Maternal Food Restriction on Lung Development and Growth of Rat Offspring
    Article Snippet: The nicotinamide adenine dinucleotide phosphate/reduced form of nicotinamide adenine dinucleotide phosphate (NADP/NADPH) assay kit was utilized to determine the levels of NADP and NADPH in the cell cultures (ab65349; Abcam, Cambridge, Massachusetts) following manufacturers’ instructions.

    Article Title: Glucose-6-phosphate dehydrogenase is critical for suppression of cardiac hypertrophy by H2S
    Article Snippet: For estimating NADP/NADPH ratio, a commercially available kit (ab65349, Abcam) was utilized, as per the manufacturer’s protocol.

    Activity Assay:

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

    Enzymatic Assay:

    Article Title: CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION
    Article Snippet: .. Because of an excess of imputed values for NAD+ and NADP+ in the Metabolon generated dataset, we measured these metabolites via commercial enzymatic assay kits (Abcam) according to the manufacturer’s instructions. .. Antibodies for western blot analysis against AMPKα1/2, phospho-AMPKα1/2 (Thr 172), rS6, and phospho-rS6 (Ser 235/236) were obtained from Cell Signaling Technologies.

    Fluorescence:

    Article Title: Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells
    Article Snippet: .. For instance, compared to MiaPaCa2.si.CTRL cells, MiaPaCa2.si.HuR cells had a rise in the NADP+ /NADPH ratio, GSSG/GSH ratio, DCF fluorescence, and mitochondrial ROS ( ). ..

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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.
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    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

    TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P

    Journal: Scientific Reports

    Article Title: TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells

    doi: 10.1038/srep35016

    Figure Lengend Snippet: TRPM2 regulated HG-induced ROS production and NADPH oxidase activation, which linked TXNIP to NLRP3 inflammasome activation in human monocytic cell lines. ( A ) ELISA for IL-1β secretion from the supernatants of treated cells. ( B,C ) The ROS production was measured by CM-F2DCFDA staining. ( D ) The level of protein carbonyl content was measured by protein carbonyl content assay. ( E ) The cellular NADPH level was measured by NADP/NADPH assay, and NADPH oxidase activity was normalized to total cellular protein levels. ( A,B ) U937 cells were stimulated with low glucose (LG; 5.5 mM glucose) or high glucose (HG; 30 mM glucose for 24, 48, 72 h) ( A ) with pre-treatment of N-acetyl-L-cysteine (NAC; 25 mM) or diphenyleneiodonium chloride (DPI; 10 μM), or ( B ) in the presence of GAPDH- (GA si), p47 phox- or TRPM2-siRNA (n = 6–7). ( C–E ) The cells were pre-treated with 3-aminobenzamide (3-AB; 5 mM), 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), DPI (10 μM), GAPDH- or TRPM2-siRNA under LG, mannitol (Ma; 30 mM) or HG conditions in ( C ) THP-1 cells or ( D,E ) U937 cells (n = 5–6). ( F ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and β-actin in the presence of GADPH- or TXNIP-siRNA under HG in U937 cells (n = 5). ( G ) Immunofluorescence images showing the location of the TXNIP and NLRP3 in fixed cells by using confocal microscopy, in the presence of GAPDH- or TRPM2-siRNA, or EGTA-AM (5 mM) under HG in U937 cells (n = 4). The percentage of TXNIP co-localization with NLRP3 inflammasome was calculated as the average volume of the overlapping areas. Data were shown as mean ± S.E.M. ( A ) *P

    Article Snippet: NADPH levels were measured by using a NADP/NADPH Assay Kit (Abcam, US), and were normalized to total cellular proteins.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Western Blot, Immunofluorescence, Confocal Microscopy

    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