Journal: Antioxidants

Article Title: Ellagic Acid Alleviates Oxidative Stress by Mediating Nrf2 Signaling Pathways and Protects against Paraquat-Induced Intestinal Injury in Piglets

doi: 10.3390/antiox11020252

Figure Lengend Snippet: Primer sequences 1 .

Article Snippet: Membranes were sealed for 10 min with a rapid blocking solution (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature and incubated for 2 h with the primary antibodies including NQO1 (1:1000; Proteintech; Chicago, IL, USA), Nrf2 (1:600; Abcam; Cambridge, UK), HO-1(1:800; Abcam; Cambridge, UK), Recombinant Kelch Like ECH Associated Protein 1 (Keap1) (1:800; Proteintech; Chicago, IL, USA), and β-actin (1:1000; Cell Signaling Technology; Danvers, MA, USA), along with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000; ZSGB, Biological Technology, Beijing, China).

Techniques:

Journal: Antioxidants

Article Title: Ellagic Acid Alleviates Oxidative Stress by Mediating Nrf2 Signaling Pathways and Protects against Paraquat-Induced Intestinal Injury in Piglets

doi: 10.3390/antiox11020252

Figure Lengend Snippet: Protein abundances of Nrf2 signaling pathway of cytosol and nucleus of jejunal and ileal mucosa in piglets. ( A ) Nuclear factor erythroid 2-related factor 2 (Nrf2), Recombinant Kelch Like ECH Associated Protein 1 (Keap1), heme oxygenase-1 (HO-1), and quinone oxidoreductase 1 (NQO1) protein abundances in cytosol of jejunal and ileal mucosa. ( B ) Nrf2, HO-1, and NQO1 protein abundances in nucleus of jejunal and ileal mucosa. PQ = 4 mg/kg paraquet; EL = 0.005% ellagic acid + 4 mg/kg paraquet; EM = 0.01% ellagic acid + 4 mg/kg paraquet; EH = 0.02% ellagic acid + 4 mg/kg paraquet. n = 8. Data are shown as mean ± SEM. * means the difference was significant when compared to the control group. ns means the difference was not significant when compared to the control group. a–c Values with different lowercase letters are significantly different among PQ, EL, EM, and EH groups ( p < 0.05).

Article Snippet: Membranes were sealed for 10 min with a rapid blocking solution (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature and incubated for 2 h with the primary antibodies including NQO1 (1:1000; Proteintech; Chicago, IL, USA), Nrf2 (1:600; Abcam; Cambridge, UK), HO-1(1:800; Abcam; Cambridge, UK), Recombinant Kelch Like ECH Associated Protein 1 (Keap1) (1:800; Proteintech; Chicago, IL, USA), and β-actin (1:1000; Cell Signaling Technology; Danvers, MA, USA), along with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000; ZSGB, Biological Technology, Beijing, China).

Techniques: Recombinant, Control

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Kaplan-Meier survival analysis of mice hydrodynamically injected with the MYC transposon system and the indicated NRF2 activating guide RNAs (sgRNA) and Cas9; B) Representative diseased livers from mice injected with MYC and sgKeap1 or sgNrf2 (targeting the Keap1 interacting ETGE domain); C) In vivo growth of murine MYC/sgKeap1 tumors transduced with Nrf2-targeted or control shRNAs; Data from HCC lines 1 & 2 were combined and plotted (n=5 each); D) Kaplan-Meier survival analysis of mice that were hydrodynamically injected with the MYC transposon system and an sgRNA/Cas9 targeting Keap1 and subsequently treated with glutathione synthesis inhibitor BSO. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S1 and Table S1.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Injection, In Vivo, Transduction, Control, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Measuring NRF2 stability using an NRF2-nanoluciferase fusion protein in lysates of KEAP1 proficient HepG2 and KEAP1 mutant Huh1 cells transduced with control or shRNA against FN3K; mean of n=6 for HepG2 and n=3 for Huh1 cells ± SD; B) Relative expression of ~80 antioxidative genes in KEAP1N414Y mutant Huh1 cells transduced with control or FN3K shRNAs; average of four replicates (n=2 for each FN3K-specific shRNAs) relative to control shRNA; C) Unsupervised clustering of total proteomics data from indicated Huh1 cell lysates; GSEA analysis of over- and underrepresented proteins in FN3K-deficient cells shows reduction of NRF2 target proteins (top) and proteins involved in xenobiotic metabolism (bottom); D) Luminescence-based quantification of oxidized and reduced glutathione in KEAP1N414Y Huh1 cells expressing control vector or shRNA against FN3K; error bar represents SD from n ≥ 5 replicates; E) Nuclear extracts from Huh1 cells with control vector or FN3K knockdown immunoprecipitated with NRF2 or IgG antibodies and probed for MAFG and β-actin; nuclear lysates (bottom) were loaded on a separate gel and probed with αNRF2 and αLamin B as indicated; F) Schematic of KEAP1-dependent and independent mechanisms of NRF2 inhibition by glycation. (*denotes two-tailed t test calculated p-value of <0.05). See also Figure S4 and Table S4.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Mutagenesis, Transduction, Control, shRNA, Expressing, Plasmid Preparation, Knockdown, Immunoprecipitation, Inhibition, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Mass spectrometric identification of tryptic peptides from unglycated (upper) and in vitro glycated recombinant NRF2 (lower); peptides that decreased in abundance upon in vitro glycation are shown in black while those that are unaffected in red; B) AUC analysis of indicated tryptic peptides generated from non-glycated and in vitro glycated NRF2; data are represented relative to R25-R34 peptide and error bar represents SD from 3 replicates; C) Spectral plot of K487-R499 peptide from unmodified (left) and in vitro glycated NRF2 (right, 5 g/L glucose for 14 days); D) Spectral intensity graphs of indicated peptides obtained from tryptic digest of immunoprecipitated nuclear NRF2 from control and FN3K deficient Huh1 cells; E) Normalized AUC analysis of indicated tryptic peptides generated by digesting immunoprecipitated NRF2 from parental (n=2) or FN3K-silenced Huh1 cells (n=3) as indicated; error bar represents SD. (* indicates p-value < 0.05). See also Figures S5, S6, and Table S5.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: In Vitro, Recombinant, Generated, Immunoprecipitation, Control

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of dual gene-targeting strategy in murine HCCs; B) Ultrasound of murine livers 4 weeks after injection with indicated plasmids; tumors are marked in red; C) Ex vivo images of livers from animals injected with indicated sgRNA combinations; D) Subcutaneous Huh1 xenografts with and without FN3K knockdown and NAC treatment as indicated measured ~30 days after implantation; E) Decreased NRF2 target protein expression by immunoblot on the FN3K deficient Huh1 tumors from panel 5D; F) Phenyl borate enrichment and immunoblot shows NRF2 glycation in the FN3K deficient Huh1 xenografts. See also Figure S7 and Table S6.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Injection, Ex Vivo, Knockdown, Expressing, Western Blot

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of our strategy for a genome-wide screen against NRF2 driven expression of the HSV-TK suicide gene; B) Map of the lentiviral vector directing ARE-controlled HSV-TK and luciferase expression; C) Change in sgRNA library representation comparing untreated cells and cells treated with the NRF2 inducer tBHQ and ganciclovir; D) Predicted sites of NRF2 protein glycation using indicated algorithms; TAD: Transactivation domain; E) Phenyl borate affinity purification and immunoblotting reveals NRF2 glycation upon FN3K knockdown in KEAP1 mutant Huh1 cells; values on top refers to % of glycated NRF2 represented by the ratio of NRF2 signal intensity in PB-bound (PB) to the sum PB-bound and flow through (FT); F) Immunoblot for nuclear (upper panel) and cytoplasmic (lower panel) levels of the indicated proteins in KEAP1 wild type HepG2 cells transduced and treated as indicated; G) Chromatin immunoprecipitation (ChIP) on indicated HepG2 nuclear lysates with anti-NRF2 antibody followed by amplification of indicated promoters; shown as % of input DNA and error bar is SD of 4 replicates; H) Viability of HepG2 cells untreated or treated with H2O2 (400 μM, 24 hours) with and with without pre-incubation with NAC (10 mM, 3 hours); mean of 9 replicates ± SD. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S3 and Table S3.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Genome Wide, Expressing, Plasmid Preparation, Luciferase, Affinity Purification, Western Blot, Knockdown, Mutagenesis, Chromatin Immunoprecipitation, Amplification, Incubation, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Unbiased, pan-cancer analysis of mutations that are significantly related (co-occurring or mutual exclusive) with KEAP1 and NRF2 using the SELECT algorithm (see text and methods for details); B) Oncoprint showing mutual exclusive relation between NRF2/ KEAP1 and EGFR mutations in human cancers; C) Nuclear extracts from HepG2 cells treated with NRF2 inducer DLS (1 μM, 24 hours), EGF (10 ng/mL, 24 hours), TGFα (1 nM, 24 hours) or DMSO and probed with antibodies against NRF2 and lamin B; D) Nuclear (upper panel) and cytoplasmic extracts (lower panel) from H3255 (EGFRL858R) cells transduced with EGFR-specific shRNA or control and immunoblotted with indicated antibodies; E) Viability of isogenic H3255 cells transduced with sgRNAs targeting KEAP1 or LacZ (control) and treated with erlotinib; error bars represent SD from 3 replicates; F) Lysates from paired PC9 cells transduced with indicated sgRNA-Cas9 constructs and treated with DMSO or erlotinib (10 nM, 6 hours) probed with the indicated antibodies. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S2 and Table S2.

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Transduction, shRNA, Control, Construct, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: GSE133160

Article Snippet: Recombinant human NRF2 , Origene , Cat#TP760529.

Techniques: Western Blot, Virus, Recombinant, Affinity Chromatography, SYBR Green Assay, Software, RNA Sequencing

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: NRF2 differentially regulates gene expression and cellular behavior in ESCC cells. Isogenic NRF2 null -KYSE70 cells were compared with NRF2 W24C -KYSE70 cells, and KEAP1 null -KYSE450 cells were compared with NRF2 WT -KYSE450 cells. ( A ) Expression of squamous differentiation and basal cell markers assessed by Western blotting. ( B ) Cell-cycle distribution determined by flow cytometry. ( C ) Cell proliferation measured by IncuCyte live-cell imaging. ( D ) Apoptosis quantified by IncuCyte analysis. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Gene Expression, Expressing, Western Blot, Flow Cytometry, Live Cell Imaging

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: PYR enhances KEAP1-dependent degradation of NRF2 W24C . ( A ) Dose-dependent reduction in NRF2 W24C protein levels in KYSE70 cells following 4 h PYR treatment. ( B ) Lack of NRF2 W24C reduction in KEAP1 null -KYSE70 cells following 4 h PYR treatment. ( C ) Co-IP analysis showing increased association between NRF2 W24C and KEAP1 following PYR treatment (10 μM, 2 h), but not between NRF2 WT and KEAP1. MTX (1 μM, 2 h) did not alter NRF2 W24C -KEAP1 association. ( D ) PLA showing increased NRF2 W24C -KEAP1 interactions following PYR treatment (10 μM, 4 h). Red dots represent PLA signal. ( E , F ) NRF2 protein levels following 72 h PYR treatment (10 μM) in KEAP1 null -KYSE70 and KEAP1 null -KYSE450 cells. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: PYR modestly enhances DLGW24C–Kelch interaction in SPR assays. SPR sensorgrams showing binding of ( A ) DLG WT peptide and ( B ) DLG W24C peptide to the KEAP1 Kelch domain. ( C ) PYR has minimal effect on DLG WT –Kelch interaction. ( D ) PYR modestly increases binding of DLG W24C peptide to the Kelch domain. The colors in an SPR sensorgrams distinguish different concentrations of the analyte (DLG peptide).

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Binding Assay

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: Computational modeling suggests PYR may facilitate NRF2 W24C –KEAP1 interaction via the Kelch domain. ( A ) Predicted interaction interface between the DLG WT motif and the Kelch domain. ( B ) Predicted weakening of the DLG W24C –Kelch interaction. ( C ) Docking of PYR into a Kelch domain pocket increases predicted interaction surface area and shape complementarity for the DLG W24C –Kelch complex. ( D ) Close-up view of PYR positioned within the Kelch domain cavity. ( E ) Two-dimensional interaction map showing predicted contacts between PYR and Kelch domain residues.

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques:

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on oxidative stress ( A ) and Nrf2 signaling pathways ( B ). ROS levels in THLE-2 cells after 24 h treatment with LPS, INT, INT+LPS, and INT+LPS+NOB (25 µM). Non-treated cells and DOXO (100 nM) served as negative and positive controls, respectively. ROS (+) and ROS (−) indicate cells with detectable or undetectable superoxide radicals, respectively. Representative histograms are shown, and data represent mean ± SEM from two independent experiments run in duplicate. Nrf2 subcellular distribution (cytosolic and nuclear fractions) and cytosolic SOD1 levels in THLE-2 cells after 24 h treatment. Representative blots are shown along with the corresponding Stain-Free™ total protein images used for total protein normalization (TPN). Lane order: (1) control, (2) LPS, (3) INT, (4) INT+LPS, (5) INT+LPS+NOB (10 µM), (6) INT+LPS+NOB (25 µM). Band intensities were quantified by densitometry and normalized to the lane-specific total protein signal obtained from Stain-Free imaging. Data are presented as mean ± SEM from two independent experiments, each performed in duplicate. Asterisks above denotes statistical significance relative to the untreated control cells with * p < 0.05, ** p < 0.01. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Target proteins were detected using primary antibodies against Nrf2 and SOD1 (Santa Cruz Biotechnology, Dallas, TX, USA), followed by AP- or HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA and BosterBio, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Staining, Control, Imaging

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Kaplan-Meier survival analysis of mice hydrodynamically injected with the MYC transposon system and the indicated NRF2 activating guide RNAs (sgRNA) and Cas9; B) Representative diseased livers from mice injected with MYC and sgKeap1 or sgNrf2 (targeting the Keap1 interacting ETGE domain); C) In vivo growth of murine MYC/sgKeap1 tumors transduced with Nrf2-targeted or control shRNAs; Data from HCC lines 1 & 2 were combined and plotted (n=5 each); D) Kaplan-Meier survival analysis of mice that were hydrodynamically injected with the MYC transposon system and an sgRNA/Cas9 targeting Keap1 and subsequently treated with glutathione synthesis inhibitor BSO. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S1 and Table S1.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Injection, In Vivo, Transduction, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Measuring NRF2 stability using an NRF2-nanoluciferase fusion protein in lysates of KEAP1 proficient HepG2 and KEAP1 mutant Huh1 cells transduced with control or shRNA against FN3K; mean of n=6 for HepG2 and n=3 for Huh1 cells ± SD; B) Relative expression of ~80 antioxidative genes in KEAP1N414Y mutant Huh1 cells transduced with control or FN3K shRNAs; average of four replicates (n=2 for each FN3K-specific shRNAs) relative to control shRNA; C) Unsupervised clustering of total proteomics data from indicated Huh1 cell lysates; GSEA analysis of over- and underrepresented proteins in FN3K-deficient cells shows reduction of NRF2 target proteins (top) and proteins involved in xenobiotic metabolism (bottom); D) Luminescence-based quantification of oxidized and reduced glutathione in KEAP1N414Y Huh1 cells expressing control vector or shRNA against FN3K; error bar represents SD from n ≥ 5 replicates; E) Nuclear extracts from Huh1 cells with control vector or FN3K knockdown immunoprecipitated with NRF2 or IgG antibodies and probed for MAFG and β-actin; nuclear lysates (bottom) were loaded on a separate gel and probed with αNRF2 and αLamin B as indicated; F) Schematic of KEAP1-dependent and independent mechanisms of NRF2 inhibition by glycation. (*denotes two-tailed t test calculated p-value of <0.05). See also Figure S4 and Table S4.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Mutagenesis, Transduction, shRNA, Expressing, Plasmid Preparation, Immunoprecipitation, Inhibition, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Mass spectrometric identification of tryptic peptides from unglycated (upper) and in vitro glycated recombinant NRF2 (lower); peptides that decreased in abundance upon in vitro glycation are shown in black while those that are unaffected in red; B) AUC analysis of indicated tryptic peptides generated from non-glycated and in vitro glycated NRF2; data are represented relative to R25-R34 peptide and error bar represents SD from 3 replicates; C) Spectral plot of K487-R499 peptide from unmodified (left) and in vitro glycated NRF2 (right, 5 g/L glucose for 14 days); D) Spectral intensity graphs of indicated peptides obtained from tryptic digest of immunoprecipitated nuclear NRF2 from control and FN3K deficient Huh1 cells; E) Normalized AUC analysis of indicated tryptic peptides generated by digesting immunoprecipitated NRF2 from parental (n=2) or FN3K-silenced Huh1 cells (n=3) as indicated; error bar represents SD. (* indicates p-value < 0.05). See also Figures S5, S6, and Table S5.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: In Vitro, Recombinant, Generated, Immunoprecipitation

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of dual gene-targeting strategy in murine HCCs; B) Ultrasound of murine livers 4 weeks after injection with indicated plasmids; tumors are marked in red; C) Ex vivo images of livers from animals injected with indicated sgRNA combinations; D) Subcutaneous Huh1 xenografts with and without FN3K knockdown and NAC treatment as indicated measured ~30 days after implantation; E) Decreased NRF2 target protein expression by immunoblot on the FN3K deficient Huh1 tumors from panel 5D; F) Phenyl borate enrichment and immunoblot shows NRF2 glycation in the FN3K deficient Huh1 xenografts. See also Figure S7 and Table S6.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Injection, Ex Vivo, Expressing, Western Blot

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of our strategy for a genome-wide screen against NRF2 driven expression of the HSV-TK suicide gene; B) Map of the lentiviral vector directing ARE-controlled HSV-TK and luciferase expression; C) Change in sgRNA library representation comparing untreated cells and cells treated with the NRF2 inducer tBHQ and ganciclovir; D) Predicted sites of NRF2 protein glycation using indicated algorithms; TAD: Transactivation domain; E) Phenyl borate affinity purification and immunoblotting reveals NRF2 glycation upon FN3K knockdown in KEAP1 mutant Huh1 cells; values on top refers to % of glycated NRF2 represented by the ratio of NRF2 signal intensity in PB-bound (PB) to the sum PB-bound and flow through (FT); F) Immunoblot for nuclear (upper panel) and cytoplasmic (lower panel) levels of the indicated proteins in KEAP1 wild type HepG2 cells transduced and treated as indicated; G) Chromatin immunoprecipitation (ChIP) on indicated HepG2 nuclear lysates with anti-NRF2 antibody followed by amplification of indicated promoters; shown as % of input DNA and error bar is SD of 4 replicates; H) Viability of HepG2 cells untreated or treated with H2O2 (400 μM, 24 hours) with and with without pre-incubation with NAC (10 mM, 3 hours); mean of 9 replicates ± SD. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S3 and Table S3.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Genome Wide, Expressing, Plasmid Preparation, Luciferase, Affinity Purification, Western Blot, Mutagenesis, Chromatin Immunoprecipitation, Amplification, Incubation, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Unbiased, pan-cancer analysis of mutations that are significantly related (co-occurring or mutual exclusive) with KEAP1 and NRF2 using the SELECT algorithm (see text and methods for details); B) Oncoprint showing mutual exclusive relation between NRF2/ KEAP1 and EGFR mutations in human cancers; C) Nuclear extracts from HepG2 cells treated with NRF2 inducer DLS (1 μM, 24 hours), EGF (10 ng/mL, 24 hours), TGFα (1 nM, 24 hours) or DMSO and probed with antibodies against NRF2 and lamin B; D) Nuclear (upper panel) and cytoplasmic extracts (lower panel) from H3255 (EGFRL858R) cells transduced with EGFR-specific shRNA or control and immunoblotted with indicated antibodies; E) Viability of isogenic H3255 cells transduced with sgRNAs targeting KEAP1 or LacZ (control) and treated with erlotinib; error bars represent SD from 3 replicates; F) Lysates from paired PC9 cells transduced with indicated sgRNA-Cas9 constructs and treated with DMSO or erlotinib (10 nM, 6 hours) probed with the indicated antibodies. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S2 and Table S2.

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Transduction, shRNA, Construct, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: In vitro glycation and PB affinity chromatography Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Western Blot, Recombinant, Affinity Chromatography, SYBR Green Assay, Software, RNA Sequencing Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Figure 2. CSE promoter activity in response to RSV infection, tBHQ stimulation and NRF2 expression. iSAE cells were transfected with -973 CSE-PGL4-Luc and Renilla-Luc plasmid (pRL-CMV) in 24-well plates. After 24 h, transfected cells were left untreated (control) or treated with tBHQ (25 µM) for 15 h or infected with RSV for 24 h alone or in combination with tBHQ (RSV + tBHQ). Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to control cells, # p < 0.05 compared to RSV infected cells (A). iSAE cells were co-transfected with increasing amounts of NRF2 expression vector or the corresponding empty vector and harvested 48 h post transfection to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to empty vector (B). Each circle represents individual data.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ Antioxidants 2022, 11, 1582 4 of 12 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGGGTCAGTGTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCCAGTCACAGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Activity Assay, Infection, Expressing, Transfection, Plasmid Preparation, Control, Luciferase

Journal: Antioxidants (Basel, Switzerland)

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Figure 3. Effects of 5’ deletions in the CSE promoter sequence on NRF2-inducible activity. Schematic representation of the CSE promoter deletion constructs. Numbering is relative to transcription initiation site (+1) (A). iSAE cells were co-transfected with the CSE-PGL4-Luc deletion mutants and Renilla-Luc plasmid plus either the empty vector or the NRF2 expression vector and harvested 48 h later to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to empty vector. * p < 0.05 relative to −973-PGL4- Luc (B). iSAE cells were co-transfected with the CSE-PGL4-Luc deletion mutants and Renilla-Luc plasmid for 24 h. Cells were left untreated or treated with tBHQ (25 µM) for 15 h before harvesting to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to control cells (C). Each circle represents individual data.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ Antioxidants 2022, 11, 1582 4 of 12 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGGGTCAGTGTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCCAGTCACAGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Sequencing, Activity Assay, Construct, Transfection, Plasmid Preparation, Expressing, Luciferase, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Figure 4. Effects of site mutations in the CSE promoter sequence on NRF2-inducible activity. Schematic representation of the CSE promoter site mutation constructs. Numbering is relative to transcription initiation site. Four segments of nine nucleotides for each of the 36 base pair regions of the CSE promoter comprised between nt −156 and −120 were mutated separately (mutation sites shown in italic underlined) (A). iSAE cells were co-transfected with the wild type (WT) −250-CSE- PGL4-Luc or the site mutants (M1 to M4) and Renilla-Luc plasmid plus either the empty vector or the NRF2 expression vector and harvested 48 h later to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to empty vector (each circle represents individual data). * p < 0.05 relative to WT PGL4-Luc (B). Nucleotide comparison of putative CSE antioxidant response element (ARE) with canonical (NQO1) and non-canonical (heme oxigenase-1, HO-1 and HSF1) ARE sites (C).

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ Antioxidants 2022, 11, 1582 4 of 12 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGGGTCAGTGTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCCAGTCACAGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Sequencing, Activity Assay, Mutagenesis, Construct, Transfection, Plasmid Preparation, Expressing, Luciferase, Comparison

Journal: Antioxidants (Basel, Switzerland)

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Figure 5. EMSA of CSE putative ARE binding complexes in response to tBHQ treatment. Nuclear extracts (6 µg) from control and SAE cells treated with tBHQ (25 µM) for 15 h were used in EMSA. Two DNA–protein complexes, C1 and C2, are detected in control cells. C2 binding is further increased by tBHQ treatment (A). Similar complex formation is observed with purified recombinant NRF2 (rNRF2) (B). Competition assay using wild type (WT) and mutant (M) oligos (50×) and nuclear extracts of tBHQ-treated SAE cells (C). Super-shift assay with specific anti-NRF2 antibody and control IgG using nuclear extracts of tBHQ-treated SAE cells (D).

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ Antioxidants 2022, 11, 1582 4 of 12 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGGGTCAGTGTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCCAGTCACAGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Binding Assay, Control, Recombinant, Competitive Binding Assay, Mutagenesis, Super-Shift Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Figure 6. ChIP assay of endogenous CSE and NQO1 promoters. Chromatin from primary SAE cells infected with RSV for 20 h or treated with 25 µM tBHQ for 15 h was immunoprecipitated with anti-NRF2 antibody or IgG as negative control. qPCR was performed with immunoprecipi- tated DNA using primers spanning either the putative ARE site of the CSE promoter (A) or the NQO1 promoter (B). Fold change was calculated compared to IgG control. Data are expressed as mean ± SEM of three or four replicate data from two independent experiments (each circle represents individual data). * p < 0.05 relative to control cells.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ Antioxidants 2022, 11, 1582 4 of 12 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGGGTCAGTGTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCCAGTCACAGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Infection, Immunoprecipitation, Negative Control, Control