nicotinamide adenine dinucleotide phosphate nadp Search Results


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  • 99
    Millipore nadp
    Effects of PIPA on physical properties, Prx2 dimerization, and <t>NADPH</t> content of RBCs stored at 4°C prior to challenge with copper/ascorbate treatment or warming to 37°C. ( A ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 3); and ( B ) percent lysed erythrocytes ( n = 3) in untreated RBC samples (circles) or samples treated (triangles) with 15% PIPA solution (sodium phosphate, inosine, sodium pyruvate, and adenine), followed by copper/ascorbate treatment for 0 (Control), 4, 24, or 48 hours at 4°C. ( C–E ) Osmotic fragility based on solution osmolality leading to 50% hemolysis in untreated RBC samples (white), samples treated with 15% PIPA solution on day 0 (light gray), ( C ) samples treated during 1 hour with 15% PIPA solution after storage (dark gray) ( n = 3), ( D ) samples treated with 30% PIPA solution on day 0 (dark gray) ( n = 6), or ( E ) samples treated with 15% PIPA solution at week 0 and week 3 (dark gray) ( n = 6), and stored at 4°C for 0, 2, 4, or 6 weeks. ( F ) Percent lysed erythrocytes ( n = 6) in untreated RBC samples (white) or samples treated with 15% PIPA solution on day 0 (gray) and stored at 4°C for 0, 2, 4, or 6 weeks. ( G ) Total <t>NADP(H)</t> and ( H ) NADPH determined by enzymatic cycling in untreated RBC samples (white) or samples treated with 15% (light gray) or 30% (dark gray) PIPA solution on day 0 and stored at 4°C for 0, 2, 4, or 6 weeks, followed by 20 hours at 37°C ( n = 6). Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. * P ≤ 0.05, repeated-measures 2-way ANOVA; # P ≤ 0.05, Mann-Whitney nonparametric test.
    Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1934 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β nadph
    NOS detection in chromatophore organs of S. officinalis . A–C , <t>NADPH</t> diaphorase reaction. A , embryo chromatophore organs. The NADPH diaphorase staining is visible in the cytoelastic sacculus, which is not fully pigmented. B , isolated juvenile chromatophore organ showing the staining in the fully pigmented sacculus and in the relaxed and contracted muscle fibers. C , isolated adult chromatophore organ with the reaction localized in contracted muscle fibers. D–F , controls of embryo, juvenile, and adult chromatophore organs with omission of <t>β-NADPH</t> showing loss of reaction and solubilization of embryo and juvenile chromatophore pigment ( D and E ). G–I , immunohistochemistry/cytochemistry reaction. G , embryo chromatophore organs. The immunopositivity is localized in the cytoelastic sacculus. H , isolated juvenile chromatophore organ showing the immunoreactivity in the pigment sacculus and in the contracted and partially relaxed muscle fibers. I , Sepia isolated adult chromatophore organ. The immunopositivity is visible in contracted and partially relaxed muscle fibers. Scale bars , 20 μm. mf , muscle fiber; sa , sacculus.
    β Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore ammonia assay kit
    NOS detection in chromatophore organs of S. officinalis . A–C , <t>NADPH</t> diaphorase reaction. A , embryo chromatophore organs. The NADPH diaphorase staining is visible in the cytoelastic sacculus, which is not fully pigmented. B , isolated juvenile chromatophore organ showing the staining in the fully pigmented sacculus and in the relaxed and contracted muscle fibers. C , isolated adult chromatophore organ with the reaction localized in contracted muscle fibers. D–F , controls of embryo, juvenile, and adult chromatophore organs with omission of <t>β-NADPH</t> showing loss of reaction and solubilization of embryo and juvenile chromatophore pigment ( D and E ). G–I , immunohistochemistry/cytochemistry reaction. G , embryo chromatophore organs. The immunopositivity is localized in the cytoelastic sacculus. H , isolated juvenile chromatophore organ showing the immunoreactivity in the pigment sacculus and in the contracted and partially relaxed muscle fibers. I , Sepia isolated adult chromatophore organ. The immunopositivity is visible in contracted and partially relaxed muscle fibers. Scale bars , 20 μm. mf , muscle fiber; sa , sacculus.
    Ammonia Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam nadp nadph assay kit
    Transcription factors that determine B cell identity regulate activity of the pentose phosphate pathway and dependency on PP2A (A) Human embryonic kidney cells (HEK-293) were transduced with a polycistronic doxycycline (Dox)-inducible vector to express the B-lineage transcription factors Pax5, Ikaros, Ebf1 and Tcf3, as confirmed by Western blot after Dox treatment for the times indicated. (B) Relative <t>NADPH/NADP</t> ratios were measured before and after 24 hours of Dox-induction of the polycistronic vector (Pax5, Ikaros, Ebf1 and Tcf3) or EV. (C) Murine myeloid leukemia cells were transduced with the inducible polycistronic vector for myeloid→B cell reprogramming. Phenotypic changes were monitored by flow cytometry 48 hours after induction of Dox and expression of Pax5, Ikaros, Ebf1 and Tcf3 was confirmed by Western blot. (E) In the same cells, protein levels for PP2A Sub A, PP2A Sub C and G6pdx were measured by Western blot. (F) Human small cell lung cancer (SCLC) and normal lung epithelial (BEAS2B) cell lines were used to compare PAX5, PP2A and G6PD expression by Western blot. (G) NADPH/NADP ratios were measured in PAX5 + and PAX5 − SLCL cell lines and upon expression of PAX5 or EV. (H) Murine Pax5 +/− Ebf1 +/− BCR-ABL1 B-ALL cells were treated with the PP2A-inhibitor LB-100 at the concentrations indicated. Dose response curves depicting relative cell viability after 72 hours are depicted. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.
    Nadp Nadph Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β nicotinamide adenine dinucleotide phosphate nadp
    Transcription factors that determine B cell identity regulate activity of the pentose phosphate pathway and dependency on PP2A (A) Human embryonic kidney cells (HEK-293) were transduced with a polycistronic doxycycline (Dox)-inducible vector to express the B-lineage transcription factors Pax5, Ikaros, Ebf1 and Tcf3, as confirmed by Western blot after Dox treatment for the times indicated. (B) Relative <t>NADPH/NADP</t> ratios were measured before and after 24 hours of Dox-induction of the polycistronic vector (Pax5, Ikaros, Ebf1 and Tcf3) or EV. (C) Murine myeloid leukemia cells were transduced with the inducible polycistronic vector for myeloid→B cell reprogramming. Phenotypic changes were monitored by flow cytometry 48 hours after induction of Dox and expression of Pax5, Ikaros, Ebf1 and Tcf3 was confirmed by Western blot. (E) In the same cells, protein levels for PP2A Sub A, PP2A Sub C and G6pdx were measured by Western blot. (F) Human small cell lung cancer (SCLC) and normal lung epithelial (BEAS2B) cell lines were used to compare PAX5, PP2A and G6PD expression by Western blot. (G) NADPH/NADP ratios were measured in PAX5 + and PAX5 − SLCL cell lines and upon expression of PAX5 or EV. (H) Murine Pax5 +/− Ebf1 +/− BCR-ABL1 B-ALL cells were treated with the PP2A-inhibitor LB-100 at the concentrations indicated. Dose response curves depicting relative cell viability after 72 hours are depicted. Data are shown as mean ± standard deviation (SD) and representative of at least three independent experiments.
    β Nicotinamide Adenine Dinucleotide Phosphate Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision nadp nadph quantification kit
    Redox homeostasis in SKOV3/DDP cells is maintained intrinsically by pairing oxidative phosphorylation with pentose phosphate pathway. (A) Cellular <t>NADPH</t> content and (B) <t>NADPH/NADP</t> + ratio, (C) GSH and (D) GSSG contents, (E) total GSH (GSH plus GSSG) level and (F) GSH/GSSG ratio determined using enzymatic assays. (G) The expression level G6PD gene was detected using reverse transcription-quantitative polymerase chain reaction. (H) G6PD protein expression level was determined using western blotting, and the enzymatic activity was analyzed using a G6PD assay kit. The data are representative of three experiments. (I) Cell viability of SKOV3 or SKOV3/DDP cells was determined using a MTT assay in the presence of G6PD inhibitors (20 μ M 6-AN or 250 μ M DHEA) with or without cisplatin for 24 h. * P
    Nadp Nadph Quantification Kit, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore thioredoxin reductase
    Figure 4. Influence of Product on Reduction using <t>Thioredoxin</t> System. Intact antibody, as measured by % Main peak in the NR CE-SDS analysis as a function of time.
    Thioredoxin Reductase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore m nadp
    GAGs from paw/ankle or knee/elbow were partial inhibitors of enzymatic activity of <t>GPI.</t> Each well contained 4.2 units/ml glucose-6-phosphate dehydrogenase, 1.0 m m <t>NADP,</t> 0.4 μg/ml (6.3 μ m ) recombinant mouse GPI, and inhibitors at various
    M Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nadp nadph quantification kit
    Nutlin inhibits or promotes PPP. ( A ) Cells were treated with vehicle or Nutlin (10 μM) and/or MK2206 (10 μM) for 24 h. Lysates were analyzed for <t>NADPH.</t> Average NADPH level from triplicate was presented with SD indicated. ( B ) Cells were transfected with control siRNA, TIGAR siRNA, or G6PD siRNA and then treated with vehicle or Nutlin (10 μM) for 24 h. Lysates were analyzed for NADPH. Average NADPH level from triplicate was presented with SD indicated (left panel). Lysates were also immunoblotted for the indicated proteins (right panel). ( C ) cells were treated with Nutlin for a 12-h period and then fluxed with D-[1,2- 13 C] glucose for 15 min. Metabolites were analyzed by LC–MS. Ribose-5-P- 13 C1 levels in vehicle-treated and Nutlin-treated cells were presented with SD indicated. There is no significant difference between vehicle and Nutlin in U2OS cells ( P = 0.22). There is significant difference between vehicle and Nutlin in MHM cells ( P = 0.008). ( D ) Metabolism of 1,2- 13 C 2 -glucose through PPP to generate NADPH and into ribose-5-P- 13 C1 is schematically presented.
    Nadp Nadph Quantification Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore thioredoxin reductase assay kit
    Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injection on <t>thioredoxin</t> reductase 1 (TR1) expression and thioredoxin reductase (TR) activity in the midbrain of mice. Seven days after MPTP treatment, the midbrain of mice was dissected out and TR1 expression levels and TR activity were evaluated. (A) Decreased level of TR1 protein was observed in MPTP-treated mice by western blot analysis. Data are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (B) TR1 mRNA level in the midbrain of mice was measured using real-time reverse transcription-PCR. A significant reduction in TR1 mRNA level was found in the MPTP-treated mice. Results are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (C) TR activity in the midbrain of the mice was evaluated using a thioredoxin reductase assay kit. MPTP decreased TR activity in the mouse midbrain. Data are expressed as mean ± SEM, and there were six mice in each group. a P
    Thioredoxin Reductase Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega nicotinamide adenine dinucleotide cell based assays
    The central carbon metabolism (CCM). The CCM combines enzymatic reactions that convert carbon sources into biomass precursors. This figure shows the five main pathways forming the CCM: glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle (TCA), lipogenesis, and beta-oxidation. Two opposite metabolic demands are at the core of the CCM: anabolic reactions, which consist in biomass synthesis, and catabolic reactions, leading to the breakdown of macromolecules for energetic use. These two aspects of <t>cell</t> metabolism are managed by biochemical oscillators, including redox couples, such as <t>nicotinamide</t> <t>adenine</t> <t>dinucleotide</t> (NAD + /NADH) and nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), and the universal energy carrier, adenine triphosphate (ATP/ADP). Transitions in CCM are reported to depend on these bio-oscillators. The NAD + /NADH ratio measures the glycolytic flux (glycolysis, PPP, and oxidative phosphorylation (OXPHOS). A high NADP + /NADPH ratio rewires glucose oxidation to the pentose phosphate pathway, whereas a low NADP + /NADPH ratio triggers lipogenesis. The ATP/(ADP + Pi) ratio senses the metabolic state of the cell and may lead to metabolic switches in the CCM.
    Nicotinamide Adenine Dinucleotide Cell Based Assays, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nadp  (Abcam)
    99
    Abcam nadp
    Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced <t>NADP</t> and <t>NAD</t> ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P
    Nadp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore β nicotinamide adenine dinucleotide phosphate sodium salt
    Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. ( a – g ) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1 i-1 ) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. ( a ) Western blot decorated with the indicated antibodies. Band intensity was normalized to β -actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. ( b ) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. ( c ) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. ( d ) Relative quantification of ROS using fluorescent dye. ( e – g ) Relative ratio of oxidized to reduced <t>NADP</t> and <t>NAD</t> ( e ), glutathione levels ( f ) and glutamine uptake ( g ). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. * P
    β Nicotinamide Adenine Dinucleotide Phosphate Sodium Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega nadp nadph glo assay kit
    PHGDH-derived serine supports the transsulfuration and folate cycles. (a) Serine metabolism via the transsulfuration and folate cycles. 13 C-labelled carbons (●) unlabelled carbons ( ○ ). Gly: Glycine, Ser: Serine, CTH: Cystathionine; HCY: Homocysteine, GSH: Glutathione, γGC: γ-glutamyl cysteine, Glu: Glutamate. (b–e) A549 cells expressing scramble (SCR), NRF2 or PHGDH shRNAs were grown in the presence of U- 13 C-glucose for 24 hours and metabolites were extracted and analysed by LC/MS. (b) Analysis of 13 C-labelling on the glycine component of glutathione. (c) Analysis of 13 C-labeling on the purine metabolite adenosine monophosphate (AMP). (d,e) Analysis of glutathione (d) and AMP (e) labelling in serine high cell lines. Cells were labelled with 13 C-glucose for 24 or 48 hours as indicated. (b,d) PHGDH-derived serine is incorporated into glutathione through the generation of glycine and cysteine. M+0 denotes no carbons labelled, M+2 denotes labelling on the glycine moiety, M+3 denotes labeling on the cysteine moiety, and M+5 denotes labeling on both glycine and cysteine. M+5 labeling was not observed. (c,e) M+5 labelling occurs following ribose-5-phosphate labelling via the pentose phosphate pathway. M+7 labelling is the result of ribose labelling plus either glycine or formyl-THF labelling in the purine ring. M+9 labelling is the result of ribose labelling plus either glycine and formyl-THF labelling in the purine ring. M+0 has no labelled carbons. (f–g) LC/MS analysis of total metabolite levels in the nucleotide (f) and transsulfuration (g) pathways. (h) <t>NADPH/NADP+</t> ratios 4 days after PHGDH knockdown. Results are the average of 3 biological replicates.
    Nadp Nadph Glo Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega nadp nadph glo assay
    Inhibition of the mevalonate pathway influences the stability of the plasma membrane. It inhibits isoprenylation of the small GTP-binding proteins and, therefore, the activity of RAS signaling. As a consequence, RAS signals via RAF into the MAPK pathway, an inhibited signaling via FLI1 and JNK (c-JUN N-terminal kinase), leads to a downregulation of DNMT1. The cross-talk of RAS with PI3K-AKT-mTOR signaling influences the expression of HDACs. Additional metabolic pathways influenced by RAS signaling are glucose uptake and the OCM, which may both be fueled by activating mutations of the P53 gene ( TP53 ) and play essential roles in DNA repair and inflammation. Similar to the inhibition of HMG-Co-A reductase, a downregulation of these pathways changes the concentration of <t>NADPH.</t> In addition, there is also a downregulation of the RHOA-ROCK signaling and the associated vitamin D degrading enzyme CYP24A1 (18) . This could induce a series of vitamin D−associated effects on fatty acid metabolism and epigenetics, for example (13) .
    Nadp Nadph Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam nadp nadph
    Cancer energy asset. Panel ( A ) represents the function of mitochondrial respiratory Complex I in CT26 and 4T1 cell lines, respectively. MTF virtually abolished Complex I activity (expressed as mU x mg −1 of proteins) that was instead left unaltered by all remaining treatments. This effect resulted in a marked decrease in the rate of both oxygen consumption (expressed as μMol O 2 x min −1 x mg −1 or proteins in Panel ( B ) and ATP synthesis (expressed as nMol x min −1 x mg −1 or proteins in Panel ( C ) through the pathway I-III-IV interrogated by pyruvate-malate administration. Despite this markedly different effect on OXPHOS, ATP:AMP ratio was significantly decreased also by CBX and siRNA (Panel D ) though to a lower degree with respect to MTF. Panels ( E , F ) display the original gels, run under the same experimental conditions for each cell line, documenting the expected response of the energy sensor mechanism that caused an increase in p-AMPK without altering total AMPK levels. The redox nature of H6PD triggered metabolism was confirmed by the decrease in NAD + availability, since NAD + /NADH ratio was selectively decreased by MTF (panel G ). By contrast, lactate release (expressed as mMol/10 6 cells over 24 hours) was induced by all interventions but scramble (Panel H ) despite an absent response of NADH levels. On the contrary, both CBX and siRNA, differently from the biguanide, increased the <t>NADP</t> + <t>/NADPH</t> ratio, without altering total coenzyme levels (measured in picoMol/10 6 cells) (Panels I , J ). (*=p
    Nadp Nadph, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nadph nadp ratio
    Proposed model of SM-FDG uptake and its relationship with <t>NADP</t> reduction to <t>NADPH.</t> The cartoon proposes the ER contribution to FDG kinetics. Pathways of glucose and FDG are depicted in black and red, respectively. G, gluconate; 6PG, 6-P-gluconate; F-2D-G, fluoro-deoxy-gluconate; F-2D-PG, fluoro-deoxy-6P-gluconate. Gluts are depicted as squares, G6P-transporter as a circle.
    Nadph Nadp Ratio, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore spinach ferredoxin nadp reductase
    cYY conversion by <t>CYP121.</t> CYP121, an electron transport chain, <t>NADPH</t> and cYY were incubated at 30 °C for various times (0, 1, 3, 10, and 30 min) then acidified and analyzed by LC-MS. Chromatograms were monitored at 214 nm and are shown for elution times from 10 to 45 min. NADPH and cYY were identified by comparison with standards. The chemical structure of P1 is shown.
    Spinach Ferredoxin Nadp Reductase, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dhfr assay kit
    ( A ) Synthetic route of <t>DSPE-PEG-Imine-MTX</t> conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. ( B ) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). ( C ) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; <t>DHFR,</t> dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.
    Dhfr Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt nadph
    ( A ) Synthetic route of <t>DSPE-PEG-Imine-MTX</t> conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. ( B ) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). ( C ) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; <t>DHFR,</t> dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.
    β Nicotinamide Adenine Dinucleotide 2 Phosphate Reduced Tetrasodium Salt Nadph, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt nadph - by Bioz Stars, 2020-09
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    88
    Olympus eu me1
    ( A ) Synthetic route of <t>DSPE-PEG-Imine-MTX</t> conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. ( B ) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). ( C ) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; <t>DHFR,</t> dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.
    Eu Me1, supplied by Olympus, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/eu me1/product/Olympus
    Average 88 stars, based on 14 article reviews
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    Image Search Results


    Effects of PIPA on physical properties, Prx2 dimerization, and NADPH content of RBCs stored at 4°C prior to challenge with copper/ascorbate treatment or warming to 37°C. ( A ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 3); and ( B ) percent lysed erythrocytes ( n = 3) in untreated RBC samples (circles) or samples treated (triangles) with 15% PIPA solution (sodium phosphate, inosine, sodium pyruvate, and adenine), followed by copper/ascorbate treatment for 0 (Control), 4, 24, or 48 hours at 4°C. ( C–E ) Osmotic fragility based on solution osmolality leading to 50% hemolysis in untreated RBC samples (white), samples treated with 15% PIPA solution on day 0 (light gray), ( C ) samples treated during 1 hour with 15% PIPA solution after storage (dark gray) ( n = 3), ( D ) samples treated with 30% PIPA solution on day 0 (dark gray) ( n = 6), or ( E ) samples treated with 15% PIPA solution at week 0 and week 3 (dark gray) ( n = 6), and stored at 4°C for 0, 2, 4, or 6 weeks. ( F ) Percent lysed erythrocytes ( n = 6) in untreated RBC samples (white) or samples treated with 15% PIPA solution on day 0 (gray) and stored at 4°C for 0, 2, 4, or 6 weeks. ( G ) Total NADP(H) and ( H ) NADPH determined by enzymatic cycling in untreated RBC samples (white) or samples treated with 15% (light gray) or 30% (dark gray) PIPA solution on day 0 and stored at 4°C for 0, 2, 4, or 6 weeks, followed by 20 hours at 37°C ( n = 6). Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. * P ≤ 0.05, repeated-measures 2-way ANOVA; # P ≤ 0.05, Mann-Whitney nonparametric test.

    Journal: JCI Insight

    Article Title: Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs

    doi: 10.1172/jci.insight.126376

    Figure Lengend Snippet: Effects of PIPA on physical properties, Prx2 dimerization, and NADPH content of RBCs stored at 4°C prior to challenge with copper/ascorbate treatment or warming to 37°C. ( A ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 3); and ( B ) percent lysed erythrocytes ( n = 3) in untreated RBC samples (circles) or samples treated (triangles) with 15% PIPA solution (sodium phosphate, inosine, sodium pyruvate, and adenine), followed by copper/ascorbate treatment for 0 (Control), 4, 24, or 48 hours at 4°C. ( C–E ) Osmotic fragility based on solution osmolality leading to 50% hemolysis in untreated RBC samples (white), samples treated with 15% PIPA solution on day 0 (light gray), ( C ) samples treated during 1 hour with 15% PIPA solution after storage (dark gray) ( n = 3), ( D ) samples treated with 30% PIPA solution on day 0 (dark gray) ( n = 6), or ( E ) samples treated with 15% PIPA solution at week 0 and week 3 (dark gray) ( n = 6), and stored at 4°C for 0, 2, 4, or 6 weeks. ( F ) Percent lysed erythrocytes ( n = 6) in untreated RBC samples (white) or samples treated with 15% PIPA solution on day 0 (gray) and stored at 4°C for 0, 2, 4, or 6 weeks. ( G ) Total NADP(H) and ( H ) NADPH determined by enzymatic cycling in untreated RBC samples (white) or samples treated with 15% (light gray) or 30% (dark gray) PIPA solution on day 0 and stored at 4°C for 0, 2, 4, or 6 weeks, followed by 20 hours at 37°C ( n = 6). Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. * P ≤ 0.05, repeated-measures 2-way ANOVA; # P ≤ 0.05, Mann-Whitney nonparametric test.

    Article Snippet: NADPH and NADP+ were detected by fluorescence (Ex = 340 nm, Em = 445 nm) and UV254nm , respectively, and quantified using authentic standards (NADPH, Sigma-Aldrich; and NADP+ ) and ChemStation (Agilent Technologies).

    Techniques: MANN-WHITNEY

    Effects of copper/ascorbate-induced oxidative stress on physical properties, Prx2 dimerization, and NADP(H) content of freshly processed RBCs. ( A ) H 2 O 2 formation by RBCs (5% hematocrit) during 24 hours at 4°C in the absence (Control) or presence of copper/ascorbate (Asc) in fresh blood samples ( n = 6). ( B ) Percent Prx2 dimerization ( n = 6) in fresh blood samples exposed to copper/ascorbate for 0 (Control), 1, 4, 24, or 48 hours. ( C ) Total NADP(H) and ( D ) NADPH by enzymatic cycling in fresh blood samples exposed to copper/ascorbate for 0, 1, 4, 24, or 48 hours at 4°C ( n = 3). ( E ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 3) and ( F ) percent lysed erythrocytes ( n = 3) in fresh blood samples exposed to copper/ascorbate for 0, 4, 24, or 48 hours at 4°C. Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. * P ≤ 0.05, repeated-measures 1-way ANOVA in comparison to control; # P ≤ 0.05, Mann-Whitney nonparametric test.

    Journal: JCI Insight

    Article Title: Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs

    doi: 10.1172/jci.insight.126376

    Figure Lengend Snippet: Effects of copper/ascorbate-induced oxidative stress on physical properties, Prx2 dimerization, and NADP(H) content of freshly processed RBCs. ( A ) H 2 O 2 formation by RBCs (5% hematocrit) during 24 hours at 4°C in the absence (Control) or presence of copper/ascorbate (Asc) in fresh blood samples ( n = 6). ( B ) Percent Prx2 dimerization ( n = 6) in fresh blood samples exposed to copper/ascorbate for 0 (Control), 1, 4, 24, or 48 hours. ( C ) Total NADP(H) and ( D ) NADPH by enzymatic cycling in fresh blood samples exposed to copper/ascorbate for 0, 1, 4, 24, or 48 hours at 4°C ( n = 3). ( E ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 3) and ( F ) percent lysed erythrocytes ( n = 3) in fresh blood samples exposed to copper/ascorbate for 0, 4, 24, or 48 hours at 4°C. Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. * P ≤ 0.05, repeated-measures 1-way ANOVA in comparison to control; # P ≤ 0.05, Mann-Whitney nonparametric test.

    Article Snippet: NADPH and NADP+ were detected by fluorescence (Ex = 340 nm, Em = 445 nm) and UV254nm , respectively, and quantified using authentic standards (NADPH, Sigma-Aldrich; and NADP+ ) and ChemStation (Agilent Technologies).

    Techniques: MANN-WHITNEY

    Effects of incubation at 37°C on physical properties, Prx2 dimerization, and NADP(H) content of RBCs previously stored at 4°C. ( A ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 6); and ( B ) elongation index (measure of RBC deformability) ( n = 6) at 0, 2, 4, and 6 weeks of storage at 4°C before (white) and after warming to 37°C for 4 hours (light gray) or 20 hours (dark gray). ( C ) Hydrogen peroxide formation as described in Methods during 20 hours at 4°C or 37°C (5% hematocrit) after storage for 0, 2, 4, or 6 weeks at 4°C ( n = 6). ( D ) Percentage Prx2 dimerization ( n = 6) at 0, 2, 4, and 6 weeks of storage at 4°C before (white) and after warming to 37°C for 4 hours or 20 hours. ( E ) Total NADP(H) and ( F ) NADPH determined by enzymatic cycling at 0, 2, 4, and 6 weeks of storage at 4°C before and after warming to 37°C for 24 hours ( n = 6). Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). * P and full uncut gels.

    Journal: JCI Insight

    Article Title: Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs

    doi: 10.1172/jci.insight.126376

    Figure Lengend Snippet: Effects of incubation at 37°C on physical properties, Prx2 dimerization, and NADP(H) content of RBCs previously stored at 4°C. ( A ) Osmotic fragility based on solution osmolality leading to 50% hemolysis ( n = 6); and ( B ) elongation index (measure of RBC deformability) ( n = 6) at 0, 2, 4, and 6 weeks of storage at 4°C before (white) and after warming to 37°C for 4 hours (light gray) or 20 hours (dark gray). ( C ) Hydrogen peroxide formation as described in Methods during 20 hours at 4°C or 37°C (5% hematocrit) after storage for 0, 2, 4, or 6 weeks at 4°C ( n = 6). ( D ) Percentage Prx2 dimerization ( n = 6) at 0, 2, 4, and 6 weeks of storage at 4°C before (white) and after warming to 37°C for 4 hours or 20 hours. ( E ) Total NADP(H) and ( F ) NADPH determined by enzymatic cycling at 0, 2, 4, and 6 weeks of storage at 4°C before and after warming to 37°C for 24 hours ( n = 6). Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). * P and full uncut gels.

    Article Snippet: NADPH and NADP+ were detected by fluorescence (Ex = 340 nm, Em = 445 nm) and UV254nm , respectively, and quantified using authentic standards (NADPH, Sigma-Aldrich; and NADP+ ) and ChemStation (Agilent Technologies).

    Techniques: Incubation

    Schematic workflow for the metabolite profiling of ketoconazole using liquid chromatography–high resolution mass spectrometry (LC-HRMS)-based metabolomics. KCZ: ketoconazole, NADPH: reduced form of nicotinamide adenine dinucleotide phosphate, KCN: potassium cyanide, MS/MS: tandem mass spectrometry, MDA: multivariate data analysis, PLS-DA: partial least squares discriminant analysis, OPLS-DA: orthogonal partial least squares discriminant analysis.

    Journal: International Journal of Molecular Sciences

    Article Title: Revisiting the Metabolism and Bioactivation of Ketoconazole in Human and Mouse Using Liquid Chromatography–Mass Spectrometry-Based Metabolomics

    doi: 10.3390/ijms18030621

    Figure Lengend Snippet: Schematic workflow for the metabolite profiling of ketoconazole using liquid chromatography–high resolution mass spectrometry (LC-HRMS)-based metabolomics. KCZ: ketoconazole, NADPH: reduced form of nicotinamide adenine dinucleotide phosphate, KCN: potassium cyanide, MS/MS: tandem mass spectrometry, MDA: multivariate data analysis, PLS-DA: partial least squares discriminant analysis, OPLS-DA: orthogonal partial least squares discriminant analysis.

    Article Snippet: Chemicals and Reagents KCZ (≥98%), Krebs–Henseleit buffer, β-nicotinamide adenine dinucleotide phosphate (NADP), the reduced form of NADP (NADPH), glucose-6-phosphate, glucose-6-phophate dehydrogenase, magnesium chloride, potassium phosphate, GSH, potassium cyanide (KCN), semicarbazide, dimethyl sulfoxide (DMSO), and methyl cellulose were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Liquid Chromatography, Mass Spectrometry, Multiple Displacement Amplification

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M109.083428

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

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

    Techniques: Staining, Isolation, Immunohistochemistry

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2018.02.048

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

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

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

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2018.02.048

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

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

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

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2018.02.048

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

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

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

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

    Journal: International Journal of Oncology

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

    doi: 10.3892/ijo.2018.4476

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

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

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

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

    Journal: Oncology Letters

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

    doi: 10.3892/ol.2019.11005

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

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

    Techniques: Inhibition, Expressing

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep11247

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

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

    Techniques: Expressing, Activity Assay, Cell Culture

    Figure 4. Influence of Product on Reduction using Thioredoxin System. Intact antibody, as measured by % Main peak in the NR CE-SDS analysis as a function of time.

    Journal: mAbs

    Article Title: Monoclonal antibody disulfide reduction during manufacturing

    doi: 10.4161/mabs.24725

    Figure Lengend Snippet: Figure 4. Influence of Product on Reduction using Thioredoxin System. Intact antibody, as measured by % Main peak in the NR CE-SDS analysis as a function of time.

    Article Snippet: The roles of thioredoxin and thioredoxin reductase (TR) have previously been described as nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular protein disulfide reductases., An in vitro lab-scale model using this complex was optimized using recombinant human thioredoxin (Sigma), NAPDH (Calbiochem), in excess and thioredoxin reductase from rat liver (Sigma).

    Techniques:

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

    Journal:

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

    doi: 10.1074/jbc.M806458200

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

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

    Techniques: Activity Assay, Recombinant

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

    Journal: Journal of Molecular Cell Biology

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

    doi: 10.1093/jmcb/mjx051

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

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

    Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy

    Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Journal: Biotechnology for Biofuels

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    doi: 10.1186/s13068-019-1356-4

    Figure Lengend Snippet: Methanol bioconversion for improved lysine synthesis in synthetic methylotrophic E. coli . Enzymes required for the assimilation of methanol into central metabolism are shown in blue: MDH methanol dehydrogenase, HPS 3-hexulose-6-phosphate synthase, and PHI 6-phospho-3-hexuloisomerase. The genes overexpressed in the lysine biosynthetic pathway are shown in green. The cofactor generation pathway was reconstructed by expressing POS5 from S. cerevisiae to convert extra NADH and generate NADPH, which is shown in red

    Article Snippet: NADH and NADPH were assayed by means of the NAD/NADH quantitation Kit and the NADP/NADPH quantitation Kit (Sigma-Aldrich).

    Techniques: Expressing

    Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Journal: Biotechnology for Biofuels

    Article Title: Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

    doi: 10.1186/s13068-019-1356-4

    Figure Lengend Snippet: Improved lysine production in synthetic methylotrophic E. coli . a Methylotrophic E. coli was engineered for the bioconversion of methanol to improve lysine production. The extra NADH from methanol consumption was engineered to generate NADPH for lysine production. b Lysine production in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. c Methanol consumption in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5. d The intracellular NADH pools in the strains BL21/ΔfrmA-ML and BL21/ΔfrmA-ML-POS5 cultivated with and without methanol. e The intracellular NADPH pools in the strains BL21/ΔfrmA-ML-POS5 cultivated with 55 mM glucose and 50 mM methanol. f Lysine production from 13 C-methanol in the strains BL21/ΔfrmA-Mdh2-Hps-Phi, BL21/ΔfrmA-ML, and BL21/ΔfrmA-ML-POS5. Error bars indicate standard error of the mean ( n = 3)

    Article Snippet: NADH and NADPH were assayed by means of the NAD/NADH quantitation Kit and the NADP/NADPH quantitation Kit (Sigma-Aldrich).

    Techniques:

    Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injection on thioredoxin reductase 1 (TR1) expression and thioredoxin reductase (TR) activity in the midbrain of mice. Seven days after MPTP treatment, the midbrain of mice was dissected out and TR1 expression levels and TR activity were evaluated. (A) Decreased level of TR1 protein was observed in MPTP-treated mice by western blot analysis. Data are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (B) TR1 mRNA level in the midbrain of mice was measured using real-time reverse transcription-PCR. A significant reduction in TR1 mRNA level was found in the MPTP-treated mice. Results are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (C) TR activity in the midbrain of the mice was evaluated using a thioredoxin reductase assay kit. MPTP decreased TR activity in the mouse midbrain. Data are expressed as mean ± SEM, and there were six mice in each group. a P

    Journal: Neural Regeneration Research

    Article Title: Downregulation of thioredoxin reductase 1 expression in the substantia nigra pars compacta of Parkinson's disease mice

    doi: 10.3969/j.issn.1673-5374.2013.35.002

    Figure Lengend Snippet: Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injection on thioredoxin reductase 1 (TR1) expression and thioredoxin reductase (TR) activity in the midbrain of mice. Seven days after MPTP treatment, the midbrain of mice was dissected out and TR1 expression levels and TR activity were evaluated. (A) Decreased level of TR1 protein was observed in MPTP-treated mice by western blot analysis. Data are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (B) TR1 mRNA level in the midbrain of mice was measured using real-time reverse transcription-PCR. A significant reduction in TR1 mRNA level was found in the MPTP-treated mice. Results are expressed as the ratio of the absorbance of the target gene to that of the GAPDH control. (C) TR activity in the midbrain of the mice was evaluated using a thioredoxin reductase assay kit. MPTP decreased TR activity in the mouse midbrain. Data are expressed as mean ± SEM, and there were six mice in each group. a P

    Article Snippet: Colorimetric assay for thioredoxin reductase activity in the midbrain of MPTP-treated mice Thioredoxin reductase activity was determined using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Injection, Expressing, Activity Assay, Mouse Assay, Western Blot, Polymerase Chain Reaction, Reductase Assay

    Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injection on the number of thioredoxin reductase 1 (TR1)-positive cells in the substantia nigra pars compacta (SNc) of mice. (A–D) Dopaminergic neurons stained for TR1 are shown in the representative images. Scale bars: Normal group: A, × 100; B, × 400. MPTP group: C, × 100; D, × 400. Positive TR1 expression is visible as brown yellow staining (arrows). (E) The number of TR1-positive cells was reduced in the MPTP group compared with the normal group. Data are expressed as mean ± SEM, and there were six mice in each group. a P

    Journal: Neural Regeneration Research

    Article Title: Downregulation of thioredoxin reductase 1 expression in the substantia nigra pars compacta of Parkinson's disease mice

    doi: 10.3969/j.issn.1673-5374.2013.35.002

    Figure Lengend Snippet: Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injection on the number of thioredoxin reductase 1 (TR1)-positive cells in the substantia nigra pars compacta (SNc) of mice. (A–D) Dopaminergic neurons stained for TR1 are shown in the representative images. Scale bars: Normal group: A, × 100; B, × 400. MPTP group: C, × 100; D, × 400. Positive TR1 expression is visible as brown yellow staining (arrows). (E) The number of TR1-positive cells was reduced in the MPTP group compared with the normal group. Data are expressed as mean ± SEM, and there were six mice in each group. a P

    Article Snippet: Colorimetric assay for thioredoxin reductase activity in the midbrain of MPTP-treated mice Thioredoxin reductase activity was determined using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Injection, Mouse Assay, Staining, Expressing

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

    Journal: Metabolites

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

    doi: 10.3390/metabo6040033

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

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

    Techniques:

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.48

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

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

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

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2017.48

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

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

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

    PHGDH-derived serine supports the transsulfuration and folate cycles. (a) Serine metabolism via the transsulfuration and folate cycles. 13 C-labelled carbons (●) unlabelled carbons ( ○ ). Gly: Glycine, Ser: Serine, CTH: Cystathionine; HCY: Homocysteine, GSH: Glutathione, γGC: γ-glutamyl cysteine, Glu: Glutamate. (b–e) A549 cells expressing scramble (SCR), NRF2 or PHGDH shRNAs were grown in the presence of U- 13 C-glucose for 24 hours and metabolites were extracted and analysed by LC/MS. (b) Analysis of 13 C-labelling on the glycine component of glutathione. (c) Analysis of 13 C-labeling on the purine metabolite adenosine monophosphate (AMP). (d,e) Analysis of glutathione (d) and AMP (e) labelling in serine high cell lines. Cells were labelled with 13 C-glucose for 24 or 48 hours as indicated. (b,d) PHGDH-derived serine is incorporated into glutathione through the generation of glycine and cysteine. M+0 denotes no carbons labelled, M+2 denotes labelling on the glycine moiety, M+3 denotes labeling on the cysteine moiety, and M+5 denotes labeling on both glycine and cysteine. M+5 labeling was not observed. (c,e) M+5 labelling occurs following ribose-5-phosphate labelling via the pentose phosphate pathway. M+7 labelling is the result of ribose labelling plus either glycine or formyl-THF labelling in the purine ring. M+9 labelling is the result of ribose labelling plus either glycine and formyl-THF labelling in the purine ring. M+0 has no labelled carbons. (f–g) LC/MS analysis of total metabolite levels in the nucleotide (f) and transsulfuration (g) pathways. (h) NADPH/NADP+ ratios 4 days after PHGDH knockdown. Results are the average of 3 biological replicates.

    Journal: Nature genetics

    Article Title: NRF2 regulates serine biosynthesis in non-small cell lung cancer

    doi: 10.1038/ng.3421

    Figure Lengend Snippet: PHGDH-derived serine supports the transsulfuration and folate cycles. (a) Serine metabolism via the transsulfuration and folate cycles. 13 C-labelled carbons (●) unlabelled carbons ( ○ ). Gly: Glycine, Ser: Serine, CTH: Cystathionine; HCY: Homocysteine, GSH: Glutathione, γGC: γ-glutamyl cysteine, Glu: Glutamate. (b–e) A549 cells expressing scramble (SCR), NRF2 or PHGDH shRNAs were grown in the presence of U- 13 C-glucose for 24 hours and metabolites were extracted and analysed by LC/MS. (b) Analysis of 13 C-labelling on the glycine component of glutathione. (c) Analysis of 13 C-labeling on the purine metabolite adenosine monophosphate (AMP). (d,e) Analysis of glutathione (d) and AMP (e) labelling in serine high cell lines. Cells were labelled with 13 C-glucose for 24 or 48 hours as indicated. (b,d) PHGDH-derived serine is incorporated into glutathione through the generation of glycine and cysteine. M+0 denotes no carbons labelled, M+2 denotes labelling on the glycine moiety, M+3 denotes labeling on the cysteine moiety, and M+5 denotes labeling on both glycine and cysteine. M+5 labeling was not observed. (c,e) M+5 labelling occurs following ribose-5-phosphate labelling via the pentose phosphate pathway. M+7 labelling is the result of ribose labelling plus either glycine or formyl-THF labelling in the purine ring. M+9 labelling is the result of ribose labelling plus either glycine and formyl-THF labelling in the purine ring. M+0 has no labelled carbons. (f–g) LC/MS analysis of total metabolite levels in the nucleotide (f) and transsulfuration (g) pathways. (h) NADPH/NADP+ ratios 4 days after PHGDH knockdown. Results are the average of 3 biological replicates.

    Article Snippet: NADPH/NADP+ ratios NADP+ and NADPH were determined with the NADP/NADPH-glo assay kit (Promega) according to the manufacturer’s protocol.

    Techniques: Derivative Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Labeling

    Inhibition of the mevalonate pathway influences the stability of the plasma membrane. It inhibits isoprenylation of the small GTP-binding proteins and, therefore, the activity of RAS signaling. As a consequence, RAS signals via RAF into the MAPK pathway, an inhibited signaling via FLI1 and JNK (c-JUN N-terminal kinase), leads to a downregulation of DNMT1. The cross-talk of RAS with PI3K-AKT-mTOR signaling influences the expression of HDACs. Additional metabolic pathways influenced by RAS signaling are glucose uptake and the OCM, which may both be fueled by activating mutations of the P53 gene ( TP53 ) and play essential roles in DNA repair and inflammation. Similar to the inhibition of HMG-Co-A reductase, a downregulation of these pathways changes the concentration of NADPH. In addition, there is also a downregulation of the RHOA-ROCK signaling and the associated vitamin D degrading enzyme CYP24A1 (18) . This could induce a series of vitamin D−associated effects on fatty acid metabolism and epigenetics, for example (13) .

    Journal: Cancer Genetics

    Article Title: Inhibition of the mevalonate pathway affects epigenetic regulation in cancer cells

    doi: 10.1016/j.cancergen.2015.03.008

    Figure Lengend Snippet: Inhibition of the mevalonate pathway influences the stability of the plasma membrane. It inhibits isoprenylation of the small GTP-binding proteins and, therefore, the activity of RAS signaling. As a consequence, RAS signals via RAF into the MAPK pathway, an inhibited signaling via FLI1 and JNK (c-JUN N-terminal kinase), leads to a downregulation of DNMT1. The cross-talk of RAS with PI3K-AKT-mTOR signaling influences the expression of HDACs. Additional metabolic pathways influenced by RAS signaling are glucose uptake and the OCM, which may both be fueled by activating mutations of the P53 gene ( TP53 ) and play essential roles in DNA repair and inflammation. Similar to the inhibition of HMG-Co-A reductase, a downregulation of these pathways changes the concentration of NADPH. In addition, there is also a downregulation of the RHOA-ROCK signaling and the associated vitamin D degrading enzyme CYP24A1 (18) . This could induce a series of vitamin D−associated effects on fatty acid metabolism and epigenetics, for example (13) .

    Article Snippet: NADP/NADPH analyses were performed directly in 96-well culture plates after 24 or 48 hours, according to the manufacturers' instructions of the NADP/NADPH Glo Assay (Promega, Madison, WI, USA).

    Techniques: Inhibition, Binding Assay, Activity Assay, Expressing, Concentration Assay

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep25092

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

    Article Snippet: NAD+ /NADH and NADP/NADPH determination The ratio between NAD+ :NADH and NADP:NADPH in cell lysates were evaluated spectrophotometrically, at 450 nm, using the NAD/NADH Assay Kit (Abcam: ab65348) and NADP/NADPH Assay Kit (Abcam: ab65349), respectively following the manufacture’s instructions.

    Techniques: Activity Assay

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

    Journal: Molecular Metabolism

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

    doi: 10.1016/j.molmet.2019.11.007

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

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

    Techniques:

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

    Journal: Molecular Metabolism

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

    doi: 10.1016/j.molmet.2019.11.007

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

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

    Techniques: Activity Assay

    cYY conversion by CYP121. CYP121, an electron transport chain, NADPH and cYY were incubated at 30 °C for various times (0, 1, 3, 10, and 30 min) then acidified and analyzed by LC-MS. Chromatograms were monitored at 214 nm and are shown for elution times from 10 to 45 min. NADPH and cYY were identified by comparison with standards. The chemical structure of P1 is shown.

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

    Article Title: Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis

    doi: 10.1073/pnas.0812191106

    Figure Lengend Snippet: cYY conversion by CYP121. CYP121, an electron transport chain, NADPH and cYY were incubated at 30 °C for various times (0, 1, 3, 10, and 30 min) then acidified and analyzed by LC-MS. Chromatograms were monitored at 214 nm and are shown for elution times from 10 to 45 min. NADPH and cYY were identified by comparison with standards. The chemical structure of P1 is shown.

    Article Snippet: Large-scale incubation of cYY with CYP121 was carried out in a 20-mL reaction mixture consisting of 0.6 mM cYY (3.9 mg), 5 μM CYP121, 1 μM spinach ferredoxin (Sigma-Aldrich), 4 units of spinach ferredoxin-NADP+ reductase (Sigma-Aldrich), 4 mM D-glucose-6-phosphate (sodium salt, Sigma-Aldrich), 20 units of yeast glucose-6-phosphate dehydrogenase (Sigma-Aldrich), 0.4 mM NADP+ , and 10 mM MgCl2 in 100 mM Tris·HCl pH 7.2.

    Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy

    ( A ) Synthetic route of DSPE-PEG-Imine-MTX conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. ( B ) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). ( C ) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.

    Journal: International Journal of Nanomedicine

    Article Title: Design of pH-sensitive methotrexate prodrug-targeted curcumin nanoparticles for efficient dual-drug delivery and combination cancer therapy

    doi: 10.2147/IJN.S152312

    Figure Lengend Snippet: ( A ) Synthetic route of DSPE-PEG-Imine-MTX conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. ( B ) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). ( C ) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.

    Article Snippet: The enzymatic activity of DHFR in the presence of MTX or MTX conjugates was determined by a DHFR assay kit (Sigma-Aldrich) based on the NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid.

    Techniques: Electron Paramagnetic Resonance, Permeability