Journal: Burns & Trauma

Article Title: Fibroblast exosomes promote wound healing and improve the quality of healed skin via miR-29a-3p-mediated KEAP1/Nrf2 pathway activation

doi: 10.1093/burnst/tkaf035

Figure Lengend Snippet: Fibroblast exosomes improve wound healing quality by reducing oxidative stress damage. ( a ) Representative images of DHE staining and statistical analysis of the fluorescence intensity of fresh frozen tissue sections from wounds in the control, GW4869 and GW4869 + Fb-exo groups on Days 7 and 28 post-wounding (scale bar = 50 μm, n = 5). ( b ) Statistical analysis of the MDA concentration in wound tissue from the control, GW4869 and GW4869 + Fb-exo groups on Days 7 and 28 post-wounding (n = 5). ( c ) Statistical analysis of the ratio of GSH/GSSG in wound tissue in the control, GW4869 and GW4869 + Fb-exo group on Days 7 and 28 post-wounding (n = 5). ( d ) Representative images of protein immunoblot bands of KEAP1 and Nrf2 in epidermal stem cells and statistical analysis (n = 3). ( e ) Statistical analysis of the MDA concentration and the GSH/GSSG ratio after epidermal stem cells were stimulated with fibroblast exosomes under oxidative stress conditions (n = 3). ( f ) Statistical analysis of cell viability after epidermal stem cells were stimulated with fibroblast exosomes under oxidative stress conditions (n = 3). ( g ) Representative flow cytometry images and statistical analysis of cell apoptosis detected by Annexin V/PI double staining of epidermal stem cells after stimulation with fibroblast exosomes under oxidative stress conditions (n = 3). Mean ± standard deviation ( * P <0.05; * * P < 0.01; * * * P <0.001; * * * * P < 0.0001; ns not statistically significant). DAPI 4′,6-Diamidino-2-phenylindole, DHE dihydroethidium, MDA malondialdehyde, GSH glutathione, GSSG glutathione Disulfide, Nrf2 nuclear factor erythroid 2-related factor 2, KEAP1 Kelch-like ECH-associated protein 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, CCK8 cell counting kit-8, PI propidium iodide

Article Snippet: KEAP1 , , TargetMol , Cat. No. TMPY-03432.

Techniques: Staining, Fluorescence, Control, Concentration Assay, Western Blot, Flow Cytometry, Double Staining, Standard Deviation, Cell Counting

Journal: Burns & Trauma

Article Title: Fibroblast exosomes promote wound healing and improve the quality of healed skin via miR-29a-3p-mediated KEAP1/Nrf2 pathway activation

doi: 10.1093/burnst/tkaf035

Figure Lengend Snippet: Fibroblast exosomes increase the capacity for cellular antioxidant stress by suppressing KEAP1 gene transcription via miR-29a-3p. ( a ) Enrichment heatmap of miRs associated with oxidative stress pathways in fibroblast-derived exosomes. ( b ) Intracellular miR-29a-3p levels in epidermal stem cells following the phagocytosis of fibroblast-derived exosomes (n = 3). ( c ) Intracellular KEAP1 mRNA expression levels in epidermal stem cells following phagocytosis of fibroblast-derived exosomes (n = 3). ( d ) Representative western blot analysis images of Nrf2 degradation in response to recombinant KEAP1 protein and fibroblast exosomes (n = 3). ( e ) Prediction of miR-29a-3p binding sites and measurement of relative luciferase activity in epidermal stem cells cotransfected with the miR-29a-3p mimic or negative control mimic and WT or Mut Keap1 vectors (n = 3). ( * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P <0.0001; ns not statistically significant). Nrf2 nuclear factor erythroid 2-related factor 2, KEAP1 Kelch-like ECH-associated protein 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, MUT mutant

Article Snippet: KEAP1 , , TargetMol , Cat. No. TMPY-03432.

Techniques: Derivative Assay, Expressing, Western Blot, Recombinant, Binding Assay, Luciferase, Activity Assay, Negative Control, Mutagenesis

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: Frontiers in Bioengineering and Biotechnology

Article Title: Hydrogen improves the efficacy of tetrandrine in the treatment of silicosis by inhibiting vascular endothelial mesenchymal transition caused by oxidative stress

doi: 10.3389/fbioe.2025.1668524

Figure Lengend Snippet: Tetrandrine concentration decreased in lung tissue and oxidative stress excessive activated in silicosis. (A,B) , HE staining and quantitative analysis of the degree of vascular stenosis of each group of mice (n = 3), scale bar, 20 μm; (C,D) , Masson staining and collagen volume fraction of each group of mice (n = 3), scale bar, 20 μm; (E,F) , Immunofluorescence and semi-quantitative of the expression of the endothelial to mesenchymal transition (EndMT)-related proteins platelet endothelial cell adhesion molecule-1 (CD31) and alfa-smooth muscle actin (α–SMA) in the lung tissues of different mouse groups (n = 3). Scale bar, 10 μm and 2 μm, Pearson’s R is the correlation coefficient—a statistical measure that indicates both the strength and direction of a linear relationship between two continuous variables, ranging from −1 (strong negative) to +1 (strong positive); (G) , The concentration of tetrandrine in lung tissue was detected by LC/MS-MS (n = 3); (H,I) , Changes of ROS levels in the lung tissues (n = 3). Scale bar, 20 μm, ROS: reactive oxygen species, DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride, Merge: ROS combines with the cell nucleus; (J) , Western blotting of Keap1 and Nrf2 expression in the lung tissues (n = 3); (K,L) , Semi-quantitative of the expression of Keap1 and Nrf2 in the lung tissues (n = 3). a P < 0.05; aa P < 0.01 vs. the control group. Data are expressed as mean ± SD.

Article Snippet: The membranes were incubated with the primary antibodies, namely, Pink1 (1:1000 dilution, Proteintech, United States, 23274-1-AP), LC3 (1:1000 dilution, Cell Signaling Technology, United States, 12741T), sequestosome 1 (SQSTM1/p62) (1:1000 dilution, Cell Signaling Technology, United States, 5114T), Parkin (1:2000 dilution, Proteintech, United States, 14060-1-AP), Kelch-like ECH-associated protein 1 (Keap1) (1:1000 dilution, Proteintech, United States, 60027-1-Ig), nuclear factor E2-related factor 2 (Nrf2) (1:2000 dilution, Proteintech, United States, 16396-1-AP), B- cell lymphoma - 2 (Bcl-2) (1:2000 dilution, Proteintech, United States, 26593-1-AP), Bcl - 2-associated X protein (Bax) (1:5000 dilution, Proteintech, United States, 50599-2-Ig), Cytochrome C (Cyto C) (1:5000, Proteintech, United States, 10993-1-AP), Caspase 3 (1:1000 dilution, Proteintech, United States, 19677-1-AP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:500000 dilution, Proteintech, United States, 60004-1-Ig).

Techniques: Concentration Assay, Staining, Immunofluorescence, Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Hydrogen improves the efficacy of tetrandrine in the treatment of silicosis by inhibiting vascular endothelial mesenchymal transition caused by oxidative stress

doi: 10.3389/fbioe.2025.1668524

Figure Lengend Snippet: Inhibiting oxidative stress can improve the concentration of tetrandrine in lung tissue in mice with silicosis. (A,B) , Changes in the ROS levels in the lung tissues (n = 3). Scale bar, 20 μm, ROS: reactive oxygen species, DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride, Merge: ROS combines with the cell nucleus; (C) , Western blotting of Keap1, Nrf2, Pink1, Parkin, Bcl-2 and Bax expression in the lung tissues (n = 3); (D,E) , Semi-quantitative of the expression of Keap1 and Nrf2 in the lung tissues of different mouse groups (n = 3); (F) , Mitochondrial injury observed under transmission electron microscopy (TEM) (n = 3). Scale bar, 2.0 (×4.0k) and 1.0 (×8.0k) μm; (G–J) , Semi-quantitative of the expression of Pink1, Parkin, Bcl-2 and Bax in the lung tissues of different mouse groups (n = 3); (K,L) , Immunofluorescence and semi-quantitative of the expression of the endothelial to mesenchymal transition (EndMT)-related proteins platelet endothelial cell adhesion molecule-1 (CD31) and alfa-smooth muscle actin (α–SMA) in the lung tissues of different mouse groups (n = 3). Scale bar, 10 μm and 2 μm; (M) , The concentration of tetrandrine in lung tissue was detected by LC/MS-MS (n = 3 biologically independent experiments). a P < 0.05; aa P < 0.01; aaa P < 0.001 vs. the control group. b P < 0.05; bb P < 0.01 vs. the model group. Data are expressed as mean ± SD.

Article Snippet: The membranes were incubated with the primary antibodies, namely, Pink1 (1:1000 dilution, Proteintech, United States, 23274-1-AP), LC3 (1:1000 dilution, Cell Signaling Technology, United States, 12741T), sequestosome 1 (SQSTM1/p62) (1:1000 dilution, Cell Signaling Technology, United States, 5114T), Parkin (1:2000 dilution, Proteintech, United States, 14060-1-AP), Kelch-like ECH-associated protein 1 (Keap1) (1:1000 dilution, Proteintech, United States, 60027-1-Ig), nuclear factor E2-related factor 2 (Nrf2) (1:2000 dilution, Proteintech, United States, 16396-1-AP), B- cell lymphoma - 2 (Bcl-2) (1:2000 dilution, Proteintech, United States, 26593-1-AP), Bcl - 2-associated X protein (Bax) (1:5000 dilution, Proteintech, United States, 50599-2-Ig), Cytochrome C (Cyto C) (1:5000, Proteintech, United States, 10993-1-AP), Caspase 3 (1:1000 dilution, Proteintech, United States, 19677-1-AP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:500000 dilution, Proteintech, United States, 60004-1-Ig).

Techniques: Concentration Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Control

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of 6S on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of 6S on expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1 in HCT-116 cells. (B) Effect of 6S on the expression of Keap1, Nrf2, and phosphorylated Nrf2 (p-Nrf2). The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after treatment of HCT-116 cells with 6S (20 μM). β-Actin was used as an internal standard. (C) Time-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM 6S for 6 h. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E) IF staining of Nrf2. HCT-116 cells were treated with 20 μM 6S for 12 or 24 h and then fixed and labeled with anti-Nrf2 and appropriate FITC-conjugated secondary antibodies. Cells were counterstained with DAPI for visualization of the nuclei. Slides were viewed using fluorescent microscopy (DAPI, blue; Nrf2, red).

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Staining, Labeling, Microscopy

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of M2 on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of M2 on the expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1. (B) Effect of M2 on the expression of Keap1, Nrf2, and p-Nrf2. The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after the treatment of HCT-116 cells with M2 (20 μM). β-Actin was used as internal standard. (C) Time-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM M2 for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM M2 for 6 h. Cytosolic and nucleic Nrf2 were determined using western blotting with the appropriate specific antibodies. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E, F) Effects of kinase inhibitors on M2-induced Nrf2 translocation and phosphorylation (E) and HMOX1 (F) expression. PD098059 (50 μM, a MEK1 inhibitor), LY294002 (50 μM, a PI3K inhibitor), or SB202190 (50 μM, a p38 inhibitor) was used to pretreat the cells for 30 min before they were exposed to 20 μM M2. After another 24 h of incubation, whole-cell lysates were prepared and assessed for Nrf2, p-Nrf2, and HMOX1 expression by western blotting. * p < 0.05.

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Phospho-proteomics, Incubation

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Cysteine Residues of Keap1 Modified by 6S as Determined by UPLC–MS/MS Analysis a

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Modification

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: