Sub-confluent HGFs were pre-treated with 6-shogaol (5 or 10 μM) for 1 h and cultured with AGEs (500 μg/mL) or BSA (500 μg/mL) for 12 h. HO-1 and NQO1 levels were determined using a Human Heme Oxygenase 1 (HO 1) Simple Step ELISA Kit (Abcam) and an NQO1 activity assay Kit (Abcam), respectively, in accordance with the manufacturer’s instructions. Briefly, the lysate from treated cells was collected in cell extraction buffer from the HO-1 kit, incubated on ice for 20 min, and centrifuged at 18,000× g for 20 min at 4 °C. HO-1 in the supernatant responded with the antibodies and then with TBS substrate, and the absorbance of the reaction solution was measured at 450 nm. HO-1 levels were normalized to the total cell protein amount, which was measured using Bio-Rad protein assay reagent (Bio-Rad). NQO1 activity in the cell lysate samples was determined by following the reduction of menadione and the simultaneous reduction of water soluble tetrazolium salts (WST1), and expressed as dicoumarol-sensitive activity using the NQO1 kit. Briefly, the treated HGFs were solubilized in the extraction buffer from the kit, incubated on ice for 15 min and centrifuged at 18,000 × g for 20 min at 4 °C. NQO1 activity in the supernatant samples was determined and expressed as a percentage of activity in the BSA group.
Nqo1 activity assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The NQO1 activity assay kit is a laboratory tool used to measure the activity of the enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). NQO1 is involved in cellular oxidation-reduction processes. The kit provides the necessary reagents and protocols to quantify NQO1 activity in biological samples.
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Lab products found in correlation
Protein assay method, by Bio-Rad (2 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Noble agar, by BD (1 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Cell death detection elisa kit, by Roche (1 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (1 mentions)
Infinite f200 microplate reader, by Tecan (1 mentions)
Prism 8, by GraphPad (1 mentions)
11 protocols using nqo1 activity assay kit
1
HO-1 and NQO1 Regulation in HGFs
2
NQO1 Activity Assay and Cell Death Analysis
3
NQO1 Activity Assay Protocol
4
Measuring Endogenous NQO1 Activity
5
Three-stage Protein QTL Analysis
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To identify the mechanism by which our GWAS-significant SNP rs1800566 influences drug response for the four drug treatments, we conducted a three-stage protein QTL (pQTL) analysis as shown in Fig 4 . For this analysis, 24 LCLs for each genotype at rs1800566 (reference = CC, heterozygous = CT, alternate = TT) were randomly selected from the 680 LCLs, for a total of 72 LCLs (sample size based on power calculations). In this analysis, we used the genotype data (from the 1000 Genomes Project), dose-response data (measured as described in the Dose-response assays section), and the NQO1 protein activity data (measured as described in the NQO1 enzyme activity assays section) in the three-stage model specified as follows:
where AUC is the area under the curve; PC1, PC2, and PC3 are the Eigenvalues from the first three principal components calculated using EigenStrat [79 (link)]; sex is an indicator variable denoting the sex of individual i; rs1800566 is the number of minor alleles at that SNP; and NQO1_protein_activity is the baseline NQO1 protein activity measured for each cell line using the NQO1 Activity Assay Kit (ab184867) from Abcam (Cambridge, UK). Details of this three-stage analysis are included in Tables D and E and the Protein QTL Analysis section inS1 Text .
where AUC is the area under the curve; PC1, PC2, and PC3 are the Eigenvalues from the first three principal components calculated using EigenStrat [79 (link)]; sex is an indicator variable denoting the sex of individual i; rs1800566 is the number of minor alleles at that SNP; and NQO1_protein_activity is the baseline NQO1 protein activity measured for each cell line using the NQO1 Activity Assay Kit (ab184867) from Abcam (Cambridge, UK). Details of this three-stage analysis are included in Tables D and E and the Protein QTL Analysis section in
6
NQO1 Enzyme Activity Assay Protocol
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NQO1 enzyme activity was performed as previously described [23 (link)]. Briefly, 2 × 107 cells of each cell line were collected, and pellets were solubilized in extraction buffer for 20 min, after which they were centrifuged at 18,000× g for 20 min at 4 °C. Supernatants were collected into Eppendorf tubes and stored at −80 °C. Samples were run according to the manufacturer’s protocol for the NQO1 activity assay kit (Abcam). Results were read at an absorbance of 440 nm every 20 s for 5 min, utilizing the Synergy-H1 Hybrid microplate reader. Plates were shaken both before and after each reading.
7
Synthesis of NQO1 Biochromatographic Stationary Phase
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The synthesis of the NQO1 biochromatographic stationary phase was done according to the published research method [21 (link)] and was shown in the second step of Fig. 1 . Synthesis process: 1 g silica gel (5 μm, 200 Å) and 1 mL MPTS were added in 100 mL DMF, and the solution system was stirred under the conditions of nitrogen protection and 60 °C for 5 h. Then, the suspension obtained from the reaction was centrifuged at 5000× g and washed with DMF 3 times. Subsequently, the obtained precipitate was stirred in 5% GMBS (dissolved in 500 mL DMSO) at room temperature for 2 h. DMSO was used to wash the MPTS/GMBS-modified silica gel for 3 times and the gel was dried in a vacuum for 48 h. Under the condition of 4 °C, 20 μg of human NQO1 recombinant protein and 40 mg MPTS/GMBS-modified silica gel were stirred in phosphate buffer solution (PBS) for 12 h, and the NQO1 recombinant protein could be immobilized in the stationary phase. The NQO1 Activity Assay Kit (ab184867, Abcam, Cambridge, MA, USA) was used to determine the biological activity of the chromatographic stationary phase, and the color development and rate (mOD/min) per sample well were recorded.![]()
Synthetic route of NQO1 biochromatographic stationary phase and screen workflow of NQO1 binding candidate components from the extract of Scutellaria baicalensis.
8
Enzymatic Activities Quantification Protocol
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Grx1 was assayed using 2-Hydroxyethyl disulfide (HEDS) as the substrate according to the method described in Raghavachari and Lou [24] (link). In brief, the cell lysate was mixed with potassium phosphate buffer (200 mM, pH 7.5) containing 0.5 mM glutathione (GSH), 0.4 U/mL glutathione reductase (GR), 0.2 mM β-nicotinamide adenine dinucleotide phosphate, reduced tetra (cyclohexylammonium) salt (NADPH), and 2 mM hydroxyethyl disulfide
(HED). The reaction was carried out at 30 °C, and the decrease in absorbance at 340 nm was monitored for 5 min and used to determine the Grx1 activity. Trx1 activity was determined using the methods of Holmgren and Bjornstedt [25] (link). The cell lysate was mixed with potassium phosphate buffer (100 mM, pH 7.0, with 2 mM EDTA) containing 0.05 U/mL thioredoxin reductase (TR), 0.2 mM NADPH, and 0.14 mM insulin. The reaction was carried out at 30 °C, and the decrease in absorbance at 340 nm was monitored for 5 min and used to determine the Trx1 activity. NQO1 activity was measured using NQO1 activity assay kit (Abcam) according to manufacturer’s instructions.
(HED). The reaction was carried out at 30 °C, and the decrease in absorbance at 340 nm was monitored for 5 min and used to determine the Grx1 activity. Trx1 activity was determined using the methods of Holmgren and Bjornstedt [25] (link). The cell lysate was mixed with potassium phosphate buffer (100 mM, pH 7.0, with 2 mM EDTA) containing 0.05 U/mL thioredoxin reductase (TR), 0.2 mM NADPH, and 0.14 mM insulin. The reaction was carried out at 30 °C, and the decrease in absorbance at 340 nm was monitored for 5 min and used to determine the Trx1 activity. NQO1 activity was measured using NQO1 activity assay kit (Abcam) according to manufacturer’s instructions.
9
Inhibitory Kinetics of Recombinant NQO1
10
Quantitative NQO1 Enzymatic Activity Assay
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NQO1 enzymatic activity was measured using the NQO1 Activity Assay Kit (ab184867) from Abcam (Cambridge, UK). Briefly, the NQO1 activity assay is based on the dicoumarol-sensitive reduction of WST-1 in the presence of menadione using 10 ug of cellular lysate protein in a 96-well plate format. Progress of the reaction was measured at 1-min intervals by measuring absorbance at 450 nm on an Infinite F200 microplate reader (Tecan Group Ltd). A 10-min endpoint reading was chosen as a time point within the linear region of the reaction. Replicates were averaged, and activity was expressed as the OD450nm following subtraction of OD450nm + dicoumarol.
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