Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Association Between P‐NRF2 and Patient Clinicopathological Factors

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques:

Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Relationship between P‐NRF2 and SUVmax. The median SUVmax values with P‐NRF2 negativity and positivity were 3.38 (IQR 2.91‐4.21) and 4.89 (IQR 3.66‐6.73), respectively ( P < 0.0001).

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques:

Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Kaplan‐Meier curves showing the survival of patients with HCC according to the expression of P‐NRF2. (A) RFS in all patients. (B) OS in all patients.

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques: Expressing

Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Univariate and Multivariate Analyses of Factors Related to RFS and OS in Patients With HCC Who Had Undergone Hepatic Resection (Cox Proportional Hazards Analysis)

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques:

Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Relationship between NRF2 and metabolism in HCC. (A) Heatmap shows distinct differences in mRNA expression in NRF2‐high cells and NRF2‐low cells. (B,C) GLUT1 , HK2 , PKLR , and PGK1 expression were assessed using quantitative real‐time PCR in NRF2‐overexpressing cells and control cells. Abbreviations: ACLY, ATP Citrate Lyase; ACO1, Aconitase 1; ACO2, Aconitase 2; ACTB, Actin Beta; AGL, Amylo‐Alpha‐1, 6‐Glucosidase, 4‐Alpha‐Glucanotransferase; ALDOB, Aldolase, Fructose‐Bisphosphate B; ALDOC, Aldolase, Fructose‐Bisphosphate C; B2M, Beta‐2‐Microglobulin; BPGM, Bisphosphoglycerate Mutase; CS, Citrate Synthase; Ctrl, control; DLAT, Dihydrolipoamide S‐Acetyltransferase; DLD, Dihydrolipoamide Dehydrogenase; DLST, Dihydrolipoamide S‐Succinyltransferase; ENO1, Enolase 1; ENO1, Enolase 1; ENO3, Enolase 3; FBP1, Fructose‐Bisphosphatase 1; FBP2, Fructose‐Bisphosphatase 2; FH, Fumarate Hydratase; G6PC, Glucose‐6‐Phosphatase Catalytic Subunit ; G6PC3, Glucose‐6‐Phosphatase Catalytic Subunit 3; G6PD, Glucose‐6‐Phosphate Dehydrogenase; GALM, Galactose Mutarotase; GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase; GBE1, 1,4‐Alpha‐Glucan Branching Enzyme 1; GCK, Glucokinase; GPI, Glucose‐6‐Phosphate Isomerase; GSK3A, Glycogen Synthase Kinase 3 Alpha; GSK3B, Glycogen Synthase Kinase 3 Beta; GYS1, Glycogen Synthase 1; GYS2, Glycogen Synthase 2; H6PD, Hexose‐6‐Phosphate Dehydrogenase/Glucose 1‐Dehydrogenase; HK3, Hexokinase 3; HPRT1, Hypoxanthine Phosphoribosyltransferase 1; IDH1, Isocitrate Dehydrogenase (NADP(+)) 1; IDH2, Isocitrate Dehydrogenase (NADP(+)) 2; IDH3A, Isocitrate Dehydrogenase (NAD(+)) 3 Catalytic Subunit Alpha; IDH3B, Isocitrate Dehydrogenase (NAD(+)) 3 Non‐Catalytic Subunit Beta; IDH3G, Isocitrate Dehydrogenase (NAD(+)) 3 Non‐Catalytic Subunit Gamma; MDH1, Malate Dehydrogenase 1; MDH1B, Malate Dehydrogenase 1B; MDH2, Malate Dehydrogenase 2; PC, Pyruvate Carboxylase; PCK1,Phosphoenolpyruvate Carboxykinase 1; PCK2, Phosphoenolpyruvate Carboxykinase 2; PDHA1, Pyruvate Dehydrogenase E1 Subunit Alpha 1; PDHB, Pyruvate Dehydrogenase E1 Subunit Beta; PDK1, Pyruvate Dehydrogenase Kinase 1; PDK2, Pyruvate Dehydrogenase Kinase 2; PDK3, Pyruvate Dehydrogenase Kinase 3; PDK4, Pyruvate Dehydrogenase Kinase 4; PDP2, Pyruvate Dehyrogenase Phosphatase Catalytic Subunit 2; PDPR, Pyruvate Dehydrogenase Phosphatase Regulatory Subunit; PFKL, Phosphofructokinase, Liver Type; PGAM2, Phosphoglycerate Mutase 2; PGK2, Phosphoglycerate Kinase 2; PGLS, 6‐Phosphogluconolactonase; PGM1, Phosphoglucomutase 1; PGM2, Phosphoglucomutase 2; PGM3, Phosphoglucomutase 3; PHKA1, Phosphorylase Kinase Regulatory Subunit Alpha 1; PHKB, Phosphorylase Kinase Regulatory Subunit Beta; PHKG1, Phosphorylase Kinase Catalytic Subunit Gamma 1; PHKG2, Phosphorylase Kinase Catalytic Subunit Gamma 2; PRPS1, Phosphoribosyl Pyrophosphate Synthetase 1; PRPS1L1, Phosphoribosyl Pyrophosphate Synthetase 1 Like 1; PRPS2, Phosphoribosyl Pyrophosphate Synthetase 2; PYGL, Glycogen Phosphorylase L; PYGM, Glycogen Phosphorylase, Muscle Associated; RBKS, Ribokinase; RPE, Ribulose‐5‐Phosphate‐3‐Epimerase; RPIA, Ribose 5‐Phosphate Isomerase A; RPLP0, Ribosomal Protein Lateral Stalk Subunit P0; SDHA, Succinate Dehydrogenase Complex Flavoprotein Subunit A; SDHB, Succinate Dehydrogenase Complex Iron Sulfur Subunit B; SDHC, Succinate Dehydrogenase Complex Subunit C; SDHD, Succinate Dehydrogenase Complex Subunit D; SUCLA2, Succinate‐CoA Ligase ADP‐Forming Subunit Beta; SUCLG1, Succinate‐CoA Ligase GDP/ADP‐Forming Subunit Alpha; SUCLG1, Succinate‐CoA Ligase GDP/ADP‐Forming Subunit Alpha; TKT, Transketolase; TPI1, Triosephosphate Isomerase 1; UGP2, UDP‐Glucose Pyrophosphorylase 2.

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Hepatology Communications

Article Title: Impact of Nuclear Factor Erythroid 2–Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

doi: 10.1002/hep4.1838

Figure Lengend Snippet: Overexpression of NRF2 increases PD‐L1 expression through HIF1α. (A) Control and NRF2‐overexpressing Hep3B and HuH7 cell lines were treated with IFN‐γ. (B) NRF2‐overexpressing cells with control and HIF1A knockdown were treated with IFN‐γ.

Article Snippet: NRF2 plasmid (NM_006164; Origene) was transfected into HCC cells using the jetPRIME kit (Polyplus Transfection).

Techniques: Over Expression, Expressing

Journal: Biomedical Reports

Article Title: Mistimed H 2 S upregulation, Nrf2 activation and antioxidant proteins levels in renal tubular epithelial cells subjected to anoxia and reoxygenation

doi: 10.3892/br.2020.1309

Figure Lengend Snippet: Expression of Nrf2 and its transcriptional targets under anoxia or reoxygenation. (A) Expression of Nrf2 and its transcriptional targets (B) SOD-3, (C) glutathione reductase, (D) ferritin H and (E) xCT increased under anoxia, and returned to baseline levels during reoxygenation. Representative blots are presented. *P<0.001 vs. Ctrl; #P<0.001 vs. Reox. Nrf2, nuclear factor erythroid 2-like 2; Ctrl, control; Reox, reoxygenation; OD, optical density; SOD-3, superoxide dismutase 3; xCT, cystine-glutamate antiporter.

Article Snippet: Primary antibodies used were specific for CBS (1:1,000; cat. no. TA338394; OriGene Technologies Inc.), CSE (1:100; cat. no. sc-374249; Santa Cruz Biotechnology, Inc.), 3-MST (1:100; cat. no. sc-376168; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), superoxide dismutase 3 (SOD3; 1:100; cat. no. sc-271170; Santa Cruz Biotechnology, Inc.), glutathione reductase (GR; 1:100; cat. no. sc-133245; Santa Cruz Biotechnology, Inc.), ferritin heavy chain (1:100; cat. no. sc-376594; Santa Cruz Biotechnology, Inc.), cystine-glutamate antiporter (xCT; 1:1,000; cat. no. ANT-111; Alomone Labs), activated cleaved-caspase-3(175) (1:500; cat. no. 7074; Cell Signaling Technology, Inc.), p53 (1:500; cat. no. 2524 Cell Signaling Technology, Inc.), p53 phosphorylated at serine 15 (p-p53) (1:500; cat. no. 9284; Cell Signaling Technology, Inc.), Bax (1:500; cat. no. 5023; Cell Signaling Technology, Inc.) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Expressing

Journal: Neural Regeneration Research

Article Title: Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

doi: 10.4103/1673-5374.187041

Figure Lengend Snippet: Effects of EA on mRNA expression levels of Bax, Bcl-2, GCS and Nrf2 in the ischemic cortex after MCAO. Bax (A), Bcl-2 (B), GCSh (D), GCSl (E) and Nrf2 (F) mRNA expression levels, as assessed by real-time PCR. The Bax/Bcl-2 ratio is shown in C. Data ( n = 7 per group in A–C, n = 5 per group in D–F) are expressed as the mean ± SEM. Statistical significance was determined using analysis of variance followed by Tukey’s multiple comparison test for multiple comparisons. * P < 0.05, ** P < 0.01, vs . sham (sham-operation) group; # P < 0.05, vs . MCAO group; † P < 0.05, vs. EA (MCAO + EA) group. MCAO: Middle cerebral artery occlusion; EA: electroacupuncture; EA plus PD98059: MCAO + EA + PD98059; GCSh: gamma-glutamylcysteine synthetase heavy subunit; GCSl: gamma-glutamylcysteine synthetase light subunit; Nrf2: nuclear factor erythroid 2-related factor 2.

Article Snippet: Then, the sections were incubated with rabbit anti-rat GCS monoclonal antibody (1:100; BA1627, Wuhan Boster Biotechnology Company, Wuhan, Hubei Province, China) or rabbit anti-rat Nrf2 monoclonal antibody(1:150; PB0327, Wuhan Boster Biotechnology Company), as the primary antibody, and then with horseradish peroxidase-conjugated goat anti-rabbit IgG, as the secondary antibody (1:100; BA1055, Wuhan Boster Biotechnology Company).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: Neural Regeneration Research

Article Title: Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

doi: 10.4103/1673-5374.187041

Figure Lengend Snippet: Effects of EA on GCS and Nrf2 immunoreactivities in the ischemic cortex. (A) Immunohistochemical staining for GCS and Nrf2 in the cortex in the different groups (× 400) ( n = 5 per group). (B, C) Mean optical densities of GCS and Nrf2-immunoreactive cells. Data are expressed as the mean ± SEM. Paired t -test was used to compare two groups, while analysis of variance followed by Tukey’s multiple comparisons test was used for multiple comparisons. ** P < 0.01, vs . sham (sham-operation) group; ## P < 0.01, vs . MCAO group; †† P < 0.01, vs . EA (MCAO + EA) group. EA: Electroacupuncture; GCS: glutamylcysteine synthetase; Nrf2: nuclear factor erythroid 2-related factor 2; MCAO: middle cerebral artery occlusion; EA plus PD98059: MCAO + EA + PD98059.

Article Snippet: Then, the sections were incubated with rabbit anti-rat GCS monoclonal antibody (1:100; BA1627, Wuhan Boster Biotechnology Company, Wuhan, Hubei Province, China) or rabbit anti-rat Nrf2 monoclonal antibody(1:150; PB0327, Wuhan Boster Biotechnology Company), as the primary antibody, and then with horseradish peroxidase-conjugated goat anti-rabbit IgG, as the secondary antibody (1:100; BA1055, Wuhan Boster Biotechnology Company).

Techniques: Immunohistochemical staining, Staining

Journal: PLOS ONE

Article Title: Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

doi: 10.1371/journal.pone.0296792

Figure Lengend Snippet: (A-E) Representative blot images and quantitative analysis of LKB1, AMPK, Nrf2, HO-1. (F-G) Representative blot images and quantitative analysis of Nuclear-Nrf2. (H-I) Representative images of Nuclear-Nrf2 and the quantitative analysis for Nuclear-Nrf2 (scale bar, 100 μm). n = 3. Data are presented as means ± S.E.M. * p < 0.05 and ** p < 0.01 vs. db/m group; Φ p < 0.05 and ΦΦ p < 0.01 vs. db/db group.

Article Snippet: Seal with 5% skim milk powder sealing solution for 2 h, adding primary antibody and incubating overnight at 4°C: Tubulin (1:10000, GTX628802) and Caspase-3(1:1000, #14220) from CST; p-AMPK (1:2000, AP0116) and HO-1(1:1000, A1346) from ABclonal; PI3K (1:1000, AF6241) from Affinity; LKB1 (1:1000, 10746-1-AP), p-LKB1 (1:5000, 80127-1-RR), AMPK (1:500, 10929-2-AP), p-AKT (1:5000, 66444-1-Ig), AKT (1:5000, 60203-2-Ig), GLUT4 (1:3000, 66846-1-Ig) from Proteintech; Bax (1:1000, GB12690) from Seville; Bcl-2 (1:1000, BA0412) and Nrf2 (1:1000, A0674) from BOSTER; Lamin-B1(1:1000, SI17-06) from Huabio.

Techniques:

Journal: PLOS ONE

Article Title: Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

doi: 10.1371/journal.pone.0296792

Figure Lengend Snippet: (A) The cell viability of H9C2 cells was treated with HG with 0, 10, 30, and 50mM for 24 and 48 h. (B) The H9C2 cells were treated with IH and HG for 24 and 48 h, and cell viability of H9C2 cells was measured. (C) The cell viability of H9C2 cells treated with IH and HG. n = 6. (D-E) Representative images of mitochondrial membrane potential and quantitative analysis of the JC-1 ratio. n = 4. (F-G) Representative images of ROS and quantitative analysis of ROS. (H-K) Representative blot images and quantitative analysis of LKB1, AMPK, and Nrf2. n = 3. Data are presented as means ± S.E.M. * p < 0.05 and ** p < 0.01 vs. CON group; Φ p < 0.05 and ΦΦ p < 0.01 vs. HG group.

Article Snippet: Seal with 5% skim milk powder sealing solution for 2 h, adding primary antibody and incubating overnight at 4°C: Tubulin (1:10000, GTX628802) and Caspase-3(1:1000, #14220) from CST; p-AMPK (1:2000, AP0116) and HO-1(1:1000, A1346) from ABclonal; PI3K (1:1000, AF6241) from Affinity; LKB1 (1:1000, 10746-1-AP), p-LKB1 (1:5000, 80127-1-RR), AMPK (1:500, 10929-2-AP), p-AKT (1:5000, 66444-1-Ig), AKT (1:5000, 60203-2-Ig), GLUT4 (1:3000, 66846-1-Ig) from Proteintech; Bax (1:1000, GB12690) from Seville; Bcl-2 (1:1000, BA0412) and Nrf2 (1:1000, A0674) from BOSTER; Lamin-B1(1:1000, SI17-06) from Huabio.

Techniques: Membrane

Journal: PLOS ONE

Article Title: Chronic intermittent hypoxia aggravated diabetic cardiomyopathy through LKB1/AMPK/Nrf2 signaling pathway

doi: 10.1371/journal.pone.0296792

Figure Lengend Snippet: (A-B) Representative images of mitochondrial membrane potential and quantitative analysis of the JC-1 ratio. (C-D) Representative images and quantitative analysis of ROS. (E-G) Representative blot images and quantitative analysis of AMPK and Nrf2. n = 3. Data are presented as means ± S.E.M. * p < 0.05 and ** p < 0.01 vs. CON group; Φ p < 0.05 and ΦΦ p < 0.01 vs. IH+HG group.

Article Snippet: Seal with 5% skim milk powder sealing solution for 2 h, adding primary antibody and incubating overnight at 4°C: Tubulin (1:10000, GTX628802) and Caspase-3(1:1000, #14220) from CST; p-AMPK (1:2000, AP0116) and HO-1(1:1000, A1346) from ABclonal; PI3K (1:1000, AF6241) from Affinity; LKB1 (1:1000, 10746-1-AP), p-LKB1 (1:5000, 80127-1-RR), AMPK (1:500, 10929-2-AP), p-AKT (1:5000, 66444-1-Ig), AKT (1:5000, 60203-2-Ig), GLUT4 (1:3000, 66846-1-Ig) from Proteintech; Bax (1:1000, GB12690) from Seville; Bcl-2 (1:1000, BA0412) and Nrf2 (1:1000, A0674) from BOSTER; Lamin-B1(1:1000, SI17-06) from Huabio.

Techniques: Membrane

Journal: Current Research in Toxicology

Article Title: Cytoprotective effects of α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, oleic acid and α-tocopherol on 7-ketocholesterol – Induced oxiapoptophagy: Major roles of PI3-K / PDK-1 / Akt signaling pathway and glutathione peroxidase activity in cell rescue

doi: 10.1016/j.crtox.2024.100153

Figure Lengend Snippet: Effects of 7-ketocholesterol with or without α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, oleic acid, and α-tocopherol on the Nrf-2 pathway. N2a cells previously cultured for 24 h were further cultured for 48 h with or without 7-ketocholesterol (7KC, 50 µM) in the presence or absence of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and oleic acid (OA) used at 25 µM or with α-tocopherol (α-toco: 400 µM), used as a positive control for cytoprotection. A. Analysis by western blotting of total Nrf2 (Nrf2) and phosphorylated Nrf2 (P-Nrf2); β-actin was used as the loading control. B. Analysis of Nrf2, NQO1 and HMOX-1 expression by RT-qPCR; the role played by the PI3-K / Akt signaling pathway on Nrf2, NQO1 and HMOX-1 expression was also evaluated in the presence of LY-294002. Significance of the differences between control (untreated cells), and vehicle (EtOH 0.5 %)-, ALA-, EPA-, DHA-, OA-, α-toco- or 7KC-treated cells; Mann–Whitney test: * p < 0.05 or less. Significance of the differences between 7KC-treated cells and (7KC + (ALA, EPA, DHA, OA or α-toco))-treated cells; Mann–Whitney test: # p < 0.05 or less. Significance of the differences between N2a cells not treated with LY-294002, and LY-294002-treated cells; Mann– Whitney test: δ p < 0.05 or less.

Article Snippet: For the analysis of oxidative stress, the following antibodies were used: Nrf2 (rabbit polyclonal antibody, CliniSciences, Nanterre, France; ref: C0279), Nrf2 (phospho Ser 40) (rabbit polyclonal antibody, CliniSciences / GeneTex, Irvine, CA, USA; ref: GTX02873), GPx1 (rabbit polyclonal antibody, CliniSciences / GeneTex; ref: GTX116040-S), SOD1 (rabbit polyclonal antibody, CliniSciences / GeneTex; ref: GTX100554-S) and catalase (goat polyclonal antibody, R&D systems, Abingdon, UK; ref: AF3398).

Techniques: Cell Culture, Positive Control, Western Blot, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 activates and rescues NRF2 in RSV infection. ( a ) Primary human small airway epithelial cells (SAECs) were treated with 1, 3, or 5 mM GYY4137 or its vehicle. Cells were harvested 17 h after treatment and total cell lysates were analyzed by Western blot with anti-NRF2 antibody. The membrane was reprobed with anti-β-actin antibody for loading control. Western blot image is one representative of three independent experiments. The graph shows the densitometric analysis of NRF2 after normalization to β-actin expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR); ( b ) SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 (or vehicle). GYY4137 was added 1 h after RSV infection. Cells were harvested 18 h post-infection (hpi) and total cell lysates were analyzed by Western blot with anti-NRF2 antibody. The membrane was reprobed with anti-β-actin antibody for loading control. Western blot image is one representative of three independent experiments. The graph shows the densitometric analysis of NRF2 after normalization to β-actin expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV); ( c ) SAECs were infected with RSV followed by treatment with 5 mM GYY4137. Cells were harvested 18 hpi and RSV proteins were detected in total cell lysates by Western blot using anti-RSV antibody. RSV proteins corresponding bands are indicated on the right. The membrane was reprobed with anti-β-actin for loading control. Western blot image is one representative of two independent experiments.

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Infection, Western Blot, Membrane, Control

Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 rescues NRF2-dependent gene expression in RSV infection. SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 and harvested 18 hpi to prepare total RNA. SOD1, catalase, NQO1, GCLC, and GCLM gene expression were quantified by RT-qPCR. Graphs show combined data from three independent experiments expressed as mean ± SEM. The results were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV).

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Gene Expression, Infection, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 restores NRF2 ubiquitination and does not affect histone deacetylase (HDAC) activity in RSV infection. ( a ) SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 and harvested 18 hpi. NRF2 was immunoprecipitated from total cell lysates with anti-NRF2 antibody and NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody. The membrane was stripped and reprobed with anti-NRF2 antibody to determine levels of immunoprecipitated NRF2. A sample of the original pre-immunoprecipitation lysate was also analyzed by Western blot to show levels of NRF2 before immunoprecipitation (input). Western blot images are one representative of three independent experiments. Graph shows densitometric analysis of NRF2 ubiquitination after normalization to immunoprecipitated NRF2 expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV); ( b ) SAECs, uninfected and infected with RSV, were treated with 5 mM GYY4137 and harvested 18 hpi. HDAC activity (class I and class II) in nuclear extracts was measured using HDAC Fluorometric Activity Assay Kit (Cayman Chemical). The HDAC activity was normalized by protein concentration. The graph shows combined data from three independent experiments expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR).

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Ubiquitin Proteomics, Histone Deacetylase Assay, Activity Assay, Infection, Immunoprecipitation, Western Blot, Membrane, Protein Concentration

Journal: Nature Communications

Article Title: Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

doi: 10.1038/ncomms13197

Figure Lengend Snippet: ( a ) The feasibility of the approach was explored using coding mRNA data before application to lncRNAs. All coding genes were tested for associations with 68 mutational events, separately in each cancer type based on a subset of 4698 samples with available mutation data. The number of associated genes are shown for each event and cancer type (red shading) using a relatively inclusive threshold ( P <0.001 and absolute log 2 expression ratio>1, Wilcoxon rank sum test), but only associations replicated in more than one tumour type were considered further (right part, blue shading). 1,121 such consistent associations (right box with arrow) were uncovered for 30 mutational events shown here, compared with two in randomized data. The cancers with the highest mutation frequencies are indicated to the left for each event (descending order). ( b ) Reduced expression of CDKN1A / p21 in TP53 mutated compared with wild type tumours (cancers with P <0.05 are shown, Wilcoxon rank sum test; colour codes from a ). * P <0.05; ** P <0.001; *** P <1e-4. ( c ) Increased expression of GCLC in NFE2L2 (NRF2) mutated compared with wild type tumours. ( d ) Validation of coding mRNAs from a found to be associated with mutations in NFE2L2 in at least two cancers ( n =65) or four cancers ( n =10), by comparison with expression changes observed upon siRNA silencing of NFE2L2 in A549 cells 24 h post transfection ( y -axis). The x -axis shows expression ratios in NFE2L2 mutated compared with wild type tumours for cancer types with association P -value less than 0.001 (vertical bars indicate, for each gene, the mean log 2 ratio across the relevant cancer types, while coloured dots show the individual cancers). Bars indicate the mean and error bars indicate s.e.m.

Article Snippet: Immunoprecipitation of the NRF2 protein was done using a rabbit monoclonal antibody targeting the C-terminus of human NRF2 (Abcam, EP1808Y) and a rabbit polyclonal NRF2 antibody (Diagenode, C15410242).

Techniques: Mutagenesis, Expressing, Transfection

Journal: Nature Communications

Article Title: Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

doi: 10.1038/ncomms13197

Figure Lengend Snippet: ( a ) A predicted NRF2 ( NFE2L2 ) binding site (ARE element), closely matching the consensus sequence , is located ∼120 bp upstream of LINC00942 (RefSeq representation shown). ENCODE data supports transcription initiation and an upstream regulatory region coinciding with the predicted site. POL2, polymerase 2 ChIP (K562 cells, HudsonAlpha data); DNase, DNase I hypersensitivity (K562 cells, Duke); FAIRE, Formaldehyde-Assisted Isolation of Regulatory Elements (K562 cells, UNC). ( b ) ChIP analysis using two different NRF2 antibodies (Ab 1, Abcam EP1808Y; Ab 2, Diagenode C15410242) or IgG ( n =3 IPs for each antibody). A 132 bp segment centred on the predicted response element ( a ), as well as two positive control sites (in NQO1 and HMOX1 ) and three negative control regions were assayed using qPCR. Signals are shown relative to IgG (fold enrichment) and s.e.m. is indicated. ( c ) A 1 kb segment encompassing the transcription start and the NRF2 site, either wild type or mutated, was cloned into a promoter-free Gaussia luciferase (GLuc) reporter plasmid (pEZX-PG04). The plasmid was co-transfected with two different NFE2L2 siRNAs or two control siRNAs ( n =3 transfections each) in A549 and H838 cells. The signal is shown normalized to secreted alkaline phosphatase (SEAP), expressed as a control reporter from the same vector. P -values from Student's t -test combining the six transfections for control or treatment siRNAs. Error bars indicate s.e.m.

Article Snippet: Immunoprecipitation of the NRF2 protein was done using a rabbit monoclonal antibody targeting the C-terminus of human NRF2 (Abcam, EP1808Y) and a rabbit polyclonal NRF2 antibody (Diagenode, C15410242).

Techniques: Binding Assay, Sequencing, Isolation, Positive Control, Negative Control, Clone Assay, Luciferase, Plasmid Preparation, Transfection

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on proteins expression of Nrf2 and HO-1 in cells cultured with H 2 O 2 . a , Immunofluorescence staining of cytoplasmic and nuclear Nrf2 in IPEC-J2 cells. The scale bar represents 50 μm. b , Western blot analysis of nuclear Nrf2, cytoplasmic Nrf2, and HO-1. Densitometric values were normalized to those of PCNA or β-actin, as appropriate; c-e , Statistical analysis of the data in B. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H 2 O 2 and 10 nmol/L PQQ for another 2 h. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Western Blot, Incubation

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on levels of Nrf2 and HO-1 in cells treated with siRNA. a Nrf2 knockdown efficiency in cells transfected with three different sequences of Nrf2 siRNA or with NC siRNA. b and c mRNA expression levels of Nrf2 and HO-1 in IPEC-J2 cells transfected with or without Nrf2 siRNA-2. d Western blot analysis of Nrf2 and HO-1 in cells transfected with or without Nrf2 siRNA-2. e Statistical analysis of the data in D. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. * denotes a significant difference ( P < 0.05) with respect to the CTRL group. # denotes a significant difference ( P < 0.05) with respect to the H 2 O 2 group. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Cell Culture, Incubation

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on levels of ROS and ROS-regulated proteins in cells treated with siRNA. a Level of ROS in IPEC-J2 cells treated with or without Nrf2 siRNA-2. b-c Ratio of Bcl-2/Bax mRNA levels and mRNA expression levels of Caspase-3 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. d Western blot analysis of cytoplasmic Bax, Bcl-2 and Caspase-3. Densitometric values were normalized to those of β-actin. e Statistical analysis of the data in D. f Statistical analysis of the Bcl-2/Bax protein ratio. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. n = 3

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Western Blot, Cell Culture, Incubation, Transfection

Journal: Journal of Animal Science and Biotechnology

Article Title: Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

doi: 10.1186/s40104-021-00595-x

Figure Lengend Snippet: Effects of PQQ on tight junctions expression and cell viability in cells treated with siRNA. a Western blot analysis of ZO-1, ZO-2, Occludin and Claudin-1 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. The densitometric values were normalized to those of β-actin. n = 3. b Viability of IPEC-J2 cells treated with or without Nrf2 siRNA-2 and incubated in the presence or absence of H 2 O 2 or PQQ. CTRL, cells cultured with basal medium; H 2 O 2 , cells cultured with 200 μmol/L H 2 O 2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H 2 O 2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H 2 O 2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H 2 O 2 for another 2 h. n = 6. * denotes a significant difference ( P < 0.05) with respect to the CTRL group. # denotes a significant difference ( P < 0.05) with respect to the H 2 O 2 group

Article Snippet: In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan).

Techniques: Expressing, Western Blot, Incubation, Cell Culture, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Impact of Extremely Low-Frequency Electromagnetic Fields on Skeletal Muscle of Sedentary Adult Mice: A Pilot Study

doi: 10.3390/ijms25189857

Figure Lengend Snippet: Anti-oxidative enzyme expression levels and activity. ( A ) Representative immunoblots of pNRF2, SOD1, SOD2, and catalase ( Left panels ) and the corresponding densitometric analyses ( Right panels ) in skeletal muscles from mice groups after 1-week exposure (0.0, 0.1, or 1.0 mT ELF-EMFs) during which they were fed a diet without or with (+NAC) NAC supplementation. ( B ) Total antioxidant status (expressed as nM Trolox equivalent/μg protein) measured by TEAC assay (see ) in the same skeletal muscle samples tested in ( A ). ( C ) Catalase enzymatic activity (expressed as µmoles H 2 O 2 /min/mL) in the same skeletal muscle samples tested in ( A ). ( D ) Representative immunoblots of pNRF2, SOD1, SOD2, and catalase ( Left panels ) and the corresponding densitometric analyses ( Right panels ) in skeletal muscle from mice groups after 5 weeks of exposure (0.0, 0.1, or 1.0 mT ELF-EMFs), during which they were fed a diet without or with (+NAC) NAC supplementation. ( E ) Total antioxidant status (expressed as nM Trolox equivalent/μg protein) measured by TEAC assay (see ) in the same skeletal muscle samples tested in ( D ). ( F ) Catalase enzymatic activity (expressed as µmoles H 2 O 2 /min/mL) in the same skeletal muscle samples tested in ( D ). All densitometric analyses were calculated as the ratio between OD × mm 2 of each band and OD × mm 2 of the corresponding loading control band (NRF2 for pNRF2, or GAPDH for SOD1, SOD2, and catalase). All data are expressed as means ± S.E.M. from six independent experiments. * p < 0.05 and ** p < 0.01 versus 0.0 mT without NAC supplementation; # p < 0.05 versus 0.0 mT with NAC supplementation.

Article Snippet: Rabbit monoclonal antibody anti-NRF2 , 1:500 , Merck Life Science S.r.l, cd SAB4501984.

Techniques: Expressing, Activity Assay, Western Blot, Muscles, Control

Journal: International Journal of Molecular Sciences

Article Title: Impact of Extremely Low-Frequency Electromagnetic Fields on Skeletal Muscle of Sedentary Adult Mice: A Pilot Study

doi: 10.3390/ijms25189857

Figure Lengend Snippet: Primary antibodies used for Western blot analyses.

Article Snippet: Rabbit monoclonal antibody anti-NRF2 , 1:500 , Merck Life Science S.r.l, cd SAB4501984.

Techniques: Western Blot, Control