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: 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: Antioxidants

Article Title: Quercetin Attenuates Quinocetone-Induced Cell Apoptosis In Vitro by Activating the P38/Nrf2/HO-1 Pathway and Inhibiting the ROS/Mitochondrial Apoptotic Pathway

doi: 10.3390/antiox11081498

Figure Lengend Snippet: Quercetin supplementation upregulated the expression of Nrf2, HO-1, p-p38, and Bcl-2 proteins and downregulated Bax proteins in human L02 cells. QCT-alone treatment was performed at final concentrations of 2.5, 5, and 7.5 μg/mL with or without quercetin at a final concentration of 30 μM for 24 h, and the expression of Bax, Bcl-2, p-p38, Nrf2, and HO-1 proteins was examined using the Western blot method. The representative images were shown (on the left) and the values of each band were quantified using Image J (on the right). All results were presented as mean ± SD ( n = 3 independent experiments). * p < 0.05, ** p < 0.01, and *** p < 0.001, compared between the two groups. QCT, quinocetone; Que, quercetin; ns, no significance.

Article Snippet: Nrf2 inhibitor brusatol was purchased from Tauto Biotech. (Shanghai, China).

Techniques: Expressing, Concentration Assay, Western Blot

Journal: Antioxidants

Article Title: Quercetin Attenuates Quinocetone-Induced Cell Apoptosis In Vitro by Activating the P38/Nrf2/HO-1 Pathway and Inhibiting the ROS/Mitochondrial Apoptotic Pathway

doi: 10.3390/antiox11081498

Figure Lengend Snippet: Inhibition of the p38 pathway attenuates the protective effect of quercetin on QCT-induced cytotoxicity in human L02 cells. A and B, the effect of p38 inhibitor treatment (i.e., SB203580 at 10 μM) on the quercetin (at 30 μM) -induced expression of Nrf2 ( A ) and HO-1 ( B ) mRNAs. ( C ), L02 cells were pretreated with Que at 30 μM or co-treated brusatol at 40 nM for 2 h, followed by treatment with or without QCT at a final concentration of 5 μg/mL for an additional 24 h. Finally, the cell viability was measured. D-F, L02 cells were pretreated with Que at 30 μM or co-treated SB203580 at 10 μM for 2 h, followed by treatment with or without QCT at a final concentration of 5 μg/mL for an additional 24 h. Finally, the release of CytC ( D ), the activation of caspase-3 ( E ), and cell viabilities ( F ) were measured, respectively. All results were presented as mean ± SD ( n = 4 independent experiments). * p < 0.05, ** p < 0.01, and *** p < 0.001, compared between the two groups. QCT, quinocetone; Que, quercetin.

Article Snippet: Nrf2 inhibitor brusatol was purchased from Tauto Biotech. (Shanghai, China).

Techniques: Inhibition, Expressing, Concentration Assay, Activation Assay

Journal: Scientific Reports

Article Title: Clusterin overexpression protects against western diet-induced obesity and NAFLD

doi: 10.1038/s41598-020-73927-y

Figure Lengend Snippet: Protective effect of clusterin in the pathology of WD-induced NAFLD. ( A ) H&E staining in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. ( B – E ) Immunostaining for F4/80, Nrf2, p-Nrf2, and α-SMA in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. ( F ) Picro-Sirius red staining in the liver of wild type, wCLU-tg, and CLU-KO mice fed CHOW or WD. * P < 0.05; ** P < 0.01; *** P < 0.001. Magnification, × 200, × 400.

Article Snippet: Nrf2 inhibitor (ML385, CAS 846557-71-9) was purchased from Axon Medchem BV (Groningen, Netherlands).

Techniques: Staining, Immunostaining

Journal: Scientific Reports

Article Title: Clusterin overexpression protects against western diet-induced obesity and NAFLD

doi: 10.1038/s41598-020-73927-y

Figure Lengend Snippet: Schematic of the protective mechanism of clusterin via preconditioning. Clusterin induces a mild inflammatory response and oxidative stress, which enhances AMPK phosphorylation and Nrf2 expression, producing a preconditioning status.

Article Snippet: Nrf2 inhibitor (ML385, CAS 846557-71-9) was purchased from Axon Medchem BV (Groningen, Netherlands).

Techniques: Phospho-proteomics, Expressing