Journal: Frontiers in Immunology

Article Title: Pasture intake protects against commercial diet-induced lipopolysaccharide production facilitated by gut microbiota through activating intestinal alkaline phosphatase enzyme in meat geese

doi: 10.3389/fimmu.2022.1041070

Figure Lengend Snippet: Effects of Artificial Pasture Grazing System on Activation of Nrf2 Pathway in Meat Geese (A) Keap1 mRNA level in cecal tissues, (B) Nrf2 mRNA level in cecal tissues, and (C) Nrf2 - regulated genes ( NQO1, Gclc, Gclm , and GSTA4 ) mRNA levels in cecal tissues, normalized by β-actin and measured by qPCR. (D) Immunofluorescence (IF) analysis using Rabbit anti-Nrf2 (bs1074R) (1:500; v/v) showing nuclear translocation of Nrf2 in the cecal tissues of meat geese. Blue: nucleus (DAPI); Green: Nrf2-staining; Cerulean blue: merge of blue and green indicating nuclear localization of Nrf2, scale bar = 20 µm. (E) HO-1 protein level (F) GSR protein level (G) T-SOD protein level (H) GSH-PX protein level (I) T-AOC protein level (J) CAT protein level and (K) MDA protein level. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences * P < 0.05, ** P < 0.01.

Article Snippet: The tissue sections were incubated for NF-κB and Nrf2 with primary antibody Rabbit Anti-NF-κB (Affinity, P65-AF5006, 1:200; v/v) or Rabbit anti-Nrf2 (Bioss, bs-1074R, 1:500; v/v) respectively, at 4°C overnight followed by secondary antibody (HRP Goat Anti-Rabbit IgG (SeraCare, 5220-0336, 1:400; v/v) with incubation at room temperature in the dark for 50 minutes.

Techniques: Activation Assay, Immunofluorescence, Translocation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Pasture intake protects against commercial diet-induced lipopolysaccharide production facilitated by gut microbiota through activating intestinal alkaline phosphatase enzyme in meat geese

doi: 10.3389/fimmu.2022.1041070

Figure Lengend Snippet: Diagram illustrating a proposed mechanism by which artificial pasture grazing system as a high dietary fiber source up-regulates intestinal ALP-producing bacteria and Nrf2 signaling pathway while downregulating LPS-producing bacteria and ROS in meat geese. The increase in intestinal ALP-producing bacteria and the activation of Nrf2 signaling pathway maintain antioxidant and anti-inflammatory mechanisms that lower LPS-producing bacteria and LPS-induced ROS generation, intestinal mucosal deterioration, gut permeability, and metabolic endotoxemia. In the first step, the intestinal ALP attack on TLR4 and let the lipid A moiety not to allow LPS to bind with TLR4 and dephosphorylate LPS by breaking TLR4/MyD88-induced ROS production. In the second step, intestinal ALP activates Nrf2 pathway which reduces oxidative stress, so that ROS could not oxidize LC8 protein and deteriorate IKB-a to activate NF-κB pathway. In this way, intestinal ALP activates anti-inflammatory cytokines and then attenuates chronic low-grade inflammation, aging phenotypes, and metabolic syndrome. BG, blood glucose; TG, triglyceride; BW, body weight; BUN, blood urea nitrogen.

Article Snippet: The tissue sections were incubated for NF-κB and Nrf2 with primary antibody Rabbit Anti-NF-κB (Affinity, P65-AF5006, 1:200; v/v) or Rabbit anti-Nrf2 (Bioss, bs-1074R, 1:500; v/v) respectively, at 4°C overnight followed by secondary antibody (HRP Goat Anti-Rabbit IgG (SeraCare, 5220-0336, 1:400; v/v) with incubation at room temperature in the dark for 50 minutes.

Techniques: Activation Assay, Permeability

Journal: Nutrients

Article Title: The Food Contaminants Nivalenol and Deoxynivalenol Induce Inflammation in Intestinal Epithelial Cells by Regulating Reactive Oxygen Species Release

doi: 10.3390/nu9121343

Figure Lengend Snippet: ( A ) Effect of NIV, DON, and their combination (NIV + DON; 5 μM), on Nrf2 nuclear translocation, evaluated using immunofluorescence assay confocal microscopy. Scale bar: 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2, respectively; ( B ) Effect of NIV; ( C ) DON; and ( D ) their combination (NIV + DON) on HO-1 expression in the IEC-6 cells, evaluated by cytofluorimetric technique. Values, mean ± s.e.m., are expressed as mean fluorescence intensity. ° Denotes p < 0.05 vs control. * Denotes p < 0.05 vs. LPS + IFN; ( E ) Flow cytometry figures show gated cells for HO-1; ( F – H ) Histograms representing the percentage of cells positive to anti-HO-1.

Article Snippet: After blocking with bovine serum albumin (BSA) and PBS, cells were incubated with rabbit anti-nitrotyrosine (Millipore, Billerica, MA, USA), rabbit anti-Nrf2 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA), or rabbit anti-phospho p65 NF-κB (Santa Cruz Biotechnologies, Dallas, TX, USA), for 1 h at 37 °C.

Techniques: Translocation Assay, Immunofluorescence, Confocal Microscopy, Expressing, Fluorescence, Control, Flow Cytometry

Journal: Journal of translational medicine

Article Title: ABI3BP promotes renal aging through Klotho-mediated ferroptosis.

doi: 10.1186/s12967-024-05300-w

Figure Lengend Snippet: Fig. 4 ABI3BP knock out alleviates ferroptosis generation in radiation-induced mice kidneys. A Detection of Fe3+ content in mouse kidney tissue using Ruslane staining. B Immunohistochemistry of GPX4, Nrf2, and Klotho protein expression in four groups of mice (n = 3, scale bar = 50 μm). C Western Blot quantifying protein levels of Nrf2, GPX4, and Klotho in four groups (n = 3). Data presented as mean ± SD. n = 3 biologically independent repeats. Two-sided Student’s t-test, data shown are mean ± SD. *P < 0.05, **P < 0.01

Article Snippet: These included rabbit anti-ABI3BP antibody (bioss, BS-6506r, 1:500), rabbit Anti- KL Polyclonal antibody (Proteintech, 28100-1-AP, 1:500), rabbit Anti- GPX4 Polyclonal antibody (Proteintech, 30388- 1-AP, 1:500), rabbit Anti- NRF2 Polyclonal antibody (Proteintech, 16396-1-AP, 1:500), rabbit Anti- Acsl4 Polyclonal antibody (Proteintech, 22401-1-AP, 1:500), and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:1000).

Techniques: Knock-Out, Staining, Immunohistochemistry, Expressing, Western Blot

Journal: Journal of translational medicine

Article Title: ABI3BP promotes renal aging through Klotho-mediated ferroptosis.

doi: 10.1186/s12967-024-05300-w

Figure Lengend Snippet: Fig. 5 ABI3BP promotes cell senescence by increasing ferroptosis production. A Detection of Fe2+ content in aged HK-2 cells using a ferrous ion fluorescence probe (scale bar = 20 μm, n = 3). B Increased ROS content in aging HK-2 cells (n = 3). C Immunofluorescence of Nrf2 and GPX4 protein expression in aging HK-2 cells (scale bar = 20 μm, n = 3). D Western Blot showing down-regulation of Nrf2, GPX4, and Klotho protein levels in aging HK-2 cells (n = 3). E MDA level of HK-2 cells after irradiation (n = 3). F Detection of Fe2+ content in HK-2 cells using a ferrous ion fluorescence probe (n = 3). G Decreased ROS production in HK-2 cells (n = 3). H Western Blot showing protein levels of Nrf2, GPX4, and Klotho in HK-2 cells (n = 3). I Malondialdehyde level of different HK-2 cells (n = 3). J the protein levels of Nrf2, GPX4 and Klotho in HK-2 cells detected by western blot (n = 3). K Quantitative RT-PCR showing the knockout and overexpression efficiency of ABI3BP in HK-2 cells (n = 3). L The number of SA-β-Gal staining positive cells in indicated groups (scale bar = 50 μm, n = 3). n = 3 biologically independent repeats. Two-sided Student’s t-test, data shown are mean ± SD. *P < 0.05, **P < 0.01

Article Snippet: These included rabbit anti-ABI3BP antibody (bioss, BS-6506r, 1:500), rabbit Anti- KL Polyclonal antibody (Proteintech, 28100-1-AP, 1:500), rabbit Anti- GPX4 Polyclonal antibody (Proteintech, 30388- 1-AP, 1:500), rabbit Anti- NRF2 Polyclonal antibody (Proteintech, 16396-1-AP, 1:500), rabbit Anti- Acsl4 Polyclonal antibody (Proteintech, 22401-1-AP, 1:500), and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:1000).

Techniques: Fluorescence, Immunofluorescence, Expressing, Western Blot, Irradiation, Quantitative RT-PCR, Knock-Out, Over Expression, Staining

Journal: Journal of translational medicine

Article Title: ABI3BP promotes renal aging through Klotho-mediated ferroptosis.

doi: 10.1186/s12967-024-05300-w

Figure Lengend Snippet: Fig. 6 ABI3BP promotes cell senescence by regulating Klotho to induce ferroptosis. A, B Co-IP analysis revealed an interaction between ABI3BP and Klotho, but with no interaction observed between ABI3BP and GAPDH or Klotho and GAPDH. C Significantly increased Fe2+ content in HK-2 cells following the detection of KD-Klotho using a ferrous ion fluorescence probe (n = 3). D KD-Klotho, the ROS production of HK-2 cells increased significantly. E Western Blot analysis detected protein levels of Nrf2, GPX4, and Acsl4 after KD-Klotho treatment in HK-2 cells (n = 3). F Quantitative RT-PCR detection the efficiency of KD-Klotho in HK-2 cells (n = 3). G CCK8 essay assessed the proliferation ability of HK-2 cells with KD-Klotho (n = 3). H The SA-β-Gal staining of HK-2 cells with KD-Klotho. I The content of MDA in HK-2 cells with KD-Kloth (n = 3). n = 3 biologically independent repeats. Two-sided Student’s t-test, data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: These included rabbit anti-ABI3BP antibody (bioss, BS-6506r, 1:500), rabbit Anti- KL Polyclonal antibody (Proteintech, 28100-1-AP, 1:500), rabbit Anti- GPX4 Polyclonal antibody (Proteintech, 30388- 1-AP, 1:500), rabbit Anti- NRF2 Polyclonal antibody (Proteintech, 16396-1-AP, 1:500), rabbit Anti- Acsl4 Polyclonal antibody (Proteintech, 22401-1-AP, 1:500), and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:1000).

Techniques: Co-Immunoprecipitation Assay, Fluorescence, Western Blot, Quantitative RT-PCR, Staining

Journal: Antioxidants

Article Title: Galangin Regulates Oxidative Stress Levels in Porcine Embryos Through Interaction with the Neh1 Domain of Nrf2

doi: 10.3390/antiox14070822

Figure Lengend Snippet: Effects of GAL supplementation on the Nrf2/Keap1 signaling pathway in porcine parthenogenetic embryos. ( A ) Western blot analysis of Nrf2 and Keap1 levels in the control and GAL-treated groups ( n = 3). ( B ) Relative protein expression levels of Nrf2 and Keap1. ( C ) Measurement of the expression of genes related to the Nrf2/Keap1 signaling pathway in control and GAL-treated embryos by quantitative real-time reverse transcription polymerase chain reaction ( n = 3). ( D ) Representative images of Nrf2 immunofluorescence staining in the control and GAL-treated groups. Scale bar = 50 µm. ( E ) Relative fluorescence intensity of Nrf2 (control: n = 24; GAL: n = 28). ( F ) Western blot analysis of Nrf2 expression levels in the nucleus and cytoplasm of cells in the control group and GAL-treated group ( n = 3). ( G ) Relative protein expression levels of Nrf2 in the nucleus and cytoplasm. Significant differences are indicated by * ( p < 0.05) and ** ( p < 0.01).

Article Snippet: Blastocysts were fixed in 4% paraformaldehyde for 30 min, washed 3 times in PBS-PVA, placed in PBS-PVA containing 0.1% Triton X-100 for 30 min, and then incubated in 5% BSA for 1 h. The blastocysts were incubated with primary antibody (rabbit anti-Nrf2 antibody (1:200, Proteintech, 16396-1-AP, Rosemont, IL, USA), rabbit anti-Keap1 antibody (1:200, Proteintech, 50,599-2-Ig, Rosemont, IL, USA)) overnight at 4 °C and then with secondary antibody (Abbkine, A23220, Wuhan, China) for 1 h in the dark.

Techniques: Western Blot, Control, Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence

Journal: Redox biology

Article Title: A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury.

doi: 10.1016/j.redox.2021.102129

Figure Lengend Snippet: Fig. 1. (A) Chemical structures and Keap1–Nrf2 binding inhibition IC50 values of the cyclometalated iridium (III) and rhodium (III) complexes 1–6, 1a–1e, 10–23, the ligands 7 and 8, and positive control 9 (ML334) evaluated in this study using a FP assay. (B) Summary of SAR for these complexes.

Article Snippet: After permeabilization with 0.1% Triton-X-100 in PBS for 20 min, followed by blocking endogenous peroxidase with 5% goat serum in PBS at room temperature for 1 h, the slides were incubated with rabbit anti-mouse Nrf2 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 ◦C, then incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Life Technologies, Carlsbad, CA, USA) for 2 h at 37 ◦C in dark.

Techniques: Binding Assay, Inhibition, Positive Control, FP Assay

Journal: Redox biology

Article Title: A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury.

doi: 10.1016/j.redox.2021.102129

Figure Lengend Snippet: Fig. 2. Effects of 1 on Nrf2 translocation. (A) ICP-MS quantification of cell uptake for 1. LO2 cells were incubated with 5 μM of 1 at different times. Error bars represent the standard deviations of the results from three independent experiments. P values were calculated using a one-way ANOVA with Tukey’s multiple comparison test. **P < 0.01 vs. 45 min group. NS (not significant, P > 0.05) vs. 45 min group. (B) ICP-MS comparison of 1 in the nucleus and cytoplasm from LO2 cells. (C) Interactions between Keap1 and Nrf2 in LO2 cells were examined by Western blotting and co-IP. This experiment was repeated twice with similar results. (D) 1 induces nuclear translocation of Nrf2 in LO2 cells. (E) Western blot analysis of Nrf2 in the nucleus and cytoplasm in LO2 cells. This experiment was repeated twice with similar results.

Article Snippet: After permeabilization with 0.1% Triton-X-100 in PBS for 20 min, followed by blocking endogenous peroxidase with 5% goat serum in PBS at room temperature for 1 h, the slides were incubated with rabbit anti-mouse Nrf2 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 ◦C, then incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Life Technologies, Carlsbad, CA, USA) for 2 h at 37 ◦C in dark.

Techniques: Translocation Assay, Incubation, Comparison, Western Blot, Co-Immunoprecipitation Assay

Journal: Redox biology

Article Title: A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury.

doi: 10.1016/j.redox.2021.102129

Figure Lengend Snippet: Fig. 3. Complex 1 engages Keap1 in cellulo. (A and B) Thermal stabilization of Keap1 and Nrf2 by 1 and densitometry analysis. Proteins were detected by Western blotting using the corresponding antibodies. Error bars represent the standard deviations of the results from three independent experiments. *P < 0.05 vs. DMSO group. (C and D) ITC thermograms showing titration of 200 μM of 1 into (C) Keap1 (20 μM) and (D) Nrf2 (20 μM). (E) Docking pose of complex 1 with Keap1 (PDB code: 4L7B) which is depicted as a space-filling representation showing carbon (yellow), nitrogen (blue), chlorine (green) atoms. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: After permeabilization with 0.1% Triton-X-100 in PBS for 20 min, followed by blocking endogenous peroxidase with 5% goat serum in PBS at room temperature for 1 h, the slides were incubated with rabbit anti-mouse Nrf2 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 ◦C, then incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Life Technologies, Carlsbad, CA, USA) for 2 h at 37 ◦C in dark.

Techniques: Western Blot, Titration

Journal: Redox biology

Article Title: A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury.

doi: 10.1016/j.redox.2021.102129

Figure Lengend Snippet: Fig. 4. Complex 1 exerts its cellular effects via blocking the Keap1–Nrf2 interaction and activating HO-1 and NQO1. (A) Detection of intracellular ROS by confocal microscopy with excitation at 488 nm. LO2 cells seeded into 6-well plates were treated with H2O2 (800 μM) for 3 h, and 1 (5 μM) or 9 (5 μM) for 8 h. Complex 1 and ML334 9 (5 μM) had no significant effect on ROS elevation and proteasomal inhibition. (B) Measurement of proteasome activity using a commercial fluorometric assay kit after 1 (5 μM) or 9 (5 μM) treatment for 8 h. MG132 was used as a positive control. (C) Effect of 1 on mitochondrial membrane potential in LO2 cells as measured via rhodamine 123 staining. Cells seeded into 6-well plates were treated with 10 μM of positive control CCCP for 1 h, 5 μM of 9, and 1 for 8 h. Cells were imaged by confocal microscopy with excitation at 488 nm. (D) Fluorescence intensity was determined using a spectrofluorometer at an excitation wavelength of 505 nm and an emission wavelength of 534 nm. (E and F) Effects of 1 and ML334 on HO-1 and NQO1 levels in LO2 cells after 6 h treatment and densitometry analysis of Western blotting results. (G and H) Keap1 siRNA treatment produces efficient target knockdown in LO2 cells. Keap1, Nrf2, HO-1, and NQO1 were blotted to control for total protein levels. SiCon: control siRNA. siKeap1: Keap1 siRNA. Error bars represent the standard deviations of the results from three independent experiments. P values were calculated using a one-way ANOVA with Tukey’s multiple comparison test. **P < 0.01 vs. DMSO group. NS (not significant, P > 0.05) vs. DMSO group.

Article Snippet: After permeabilization with 0.1% Triton-X-100 in PBS for 20 min, followed by blocking endogenous peroxidase with 5% goat serum in PBS at room temperature for 1 h, the slides were incubated with rabbit anti-mouse Nrf2 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 ◦C, then incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Life Technologies, Carlsbad, CA, USA) for 2 h at 37 ◦C in dark.

Techniques: Blocking Assay, Confocal Microscopy, Inhibition, Activity Assay, Positive Control, Membrane, Staining, Fluorescence, Western Blot, Knockdown, Control, Comparison

Journal: Redox biology

Article Title: A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury.

doi: 10.1016/j.redox.2021.102129

Figure Lengend Snippet: Fig. 5. Complex 1 alleviates APAP-induced acute liver injury in mice. (A) Representative hematoxylin and eosin (H&E) staining of liver tissue sections. (B) Serum levels of alanine aspartate transaminase (AST) and aminotransferase (ALT). 1 induces nuclear translocation of Nrf2. Immunofluorescence staining of Nrf2 (C) and Western blot analysis of Nrf2 in the nucleus (D). (E) Effect of 1 and ML334 on the HO-1 and NQO1 levels by Western Blotting. (F) 1 reduced APAP-induced liver injury was involved in the upregulation of Nrf2-mediated antioxidative protein. Immunoblots analysis of nuclear Nrf2 (A) and Keap1 (B) expressions respectively, and Nrf2 downstream target proteins NQO1 (C) and HO-1 (D). Lamin B was used as the loading control. (G) Complex 1 alleviated APAP-induced hepatic oxidative stress. Hepatic levels of MDA, GSH, GSSG, GSH/GSSG, and enzyme activities of CAT and SOD were determined after the APAP challenge for 6 h. P values were calculated using a one-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD (n = 3 mice). ##P < 0.01 vs. Control (CON) group. *P < 0.05, **P < 0.01 vs. APAP-induced model group.

Article Snippet: After permeabilization with 0.1% Triton-X-100 in PBS for 20 min, followed by blocking endogenous peroxidase with 5% goat serum in PBS at room temperature for 1 h, the slides were incubated with rabbit anti-mouse Nrf2 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4 ◦C, then incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:200, Life Technologies, Carlsbad, CA, USA) for 2 h at 37 ◦C in dark.

Techniques: Staining, Translocation Assay, Immunofluorescence, Western Blot, Control, Comparison

Journal: Brain Sciences

Article Title: Daytime-Restricted Feeding Ameliorates Oxidative Stress by Increasing NRF2 Transcriptional Factor in the Rat Hippocampus in the Pilocarpine-Induced Acute Seizure Model

doi: 10.3390/brainsci13101442

Figure Lengend Snippet: Effect of daytime-restricted feeding on the Nrf2 protein content and the Nrf2 and GFAP fluorescence in the CA1 and CA3 subfields of the dorsal hippocampus after SE induction. Representative immunoblots of Nrf2 in the AL, DRF, ALSE, and DRFSE experimental groups in the hippocampal homogenates ( A ). Quantification of the optical density of Nrf2 protein contents in hippocampal homogenates ( B ) Representative images of double immunofluorescence for Nrf2 (red) and GFAP (green) and counterstained with Hoechst (blue) in all experimental groups in the CA1 and CA3 subfields ( C , E ). Quantification of the relative intensity of Nrf2 in the CA1 subfield of all groups ( D ). Quantification of Nrf2 relative intensity in the CA3 subfield ( F ). Data are presented as the mean ± S.D, (n = 5 rats per group). Arrows show the cytoplasmic and head arrows show nuclear the distribution of Nrf2, respectively. One-way ANOVA followed by Tukey’s multiple comparison test, *** p < 0.001, **** p < 0.0001. Scale bars: 20 μm; ML: molecular layer.

Article Snippet: The blots were probed with anti-rabbit polyclonal Nrf2 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, SC-722), and anti-rabbit polyclonal manganese superoxide dismutase (SOD2) (1:1000, Boster Biological Technology, Pleasanton, CA, USA, A00349) in TBST at 4 °C for 48 h. After three rinses with TBST for five minutes each, the membranes were incubated with anti-rabbit (1:10,000 Cell Signaling Technology, Beverly, MA, USA) or anti-mouse IgG secondary antibodies (1:5000, Santa Cruz Biotechnology, SC-516102) for 90 min at room temperature, followed by three rinses with TBST for five minutes each.

Techniques: Fluorescence, Western Blot, Immunofluorescence, Comparison

Journal: Frontiers in Pharmacology

Article Title: Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model

doi: 10.3389/fphar.2022.1033520

Figure Lengend Snippet: Urate crystal induced generation of reactive oxygen species (ROS) and its relationship to protein phosphatase 2A (PP2A) and xanthine oxidase (XO) activities, and nuclear factor erythroid 2-related factor 2 (Nrf2) expression in response to PP2A activation or XO inhibition in murine macrophages. Murine bone marrow derived macrophages (BMDMs) were primed with Pam3CSK4 (100 ng/ml) for 24 h. BMDMs were then incubated with febuxostat (February; 200 μM) or fingolimod (Fing; 2.5 μM) for 3 h followed by monosodium urate (MSU) crystals (250 μg/ml), and all assays were performed at 3 h. N-acetylcysteine (NAC) and okadaic acid (OKA) treatments were performed at 10 μM and 2.5nM, respectively. Nrf2 expression was quantified at the gene level by qPCR and at the protein level using immunofluorescence and estimating corrected total cell fluorescence (CTCF), normalized to control values. Data points represent independent experiments with two technical replicates per group. In each experiment, BMDMs were generated from the bone marrows of 2-3 animals. In the immunofluorescence studies, data points represent independent experiments with one technical replicate per group using murine J774 macrophage cell line. Control group represents untreated BMDMs or J774 macrophages. Statistical analysis was performed by ANOVA. ns, non-significant; *p < 0.05 ; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. (A) Fingolimod reduced ROS generation in MSU stimulated BMDMs. Febuxostat and NAC reduced ROS generation to a similar extent to fingolimod. (B) NAC enhanced PP2A activity in MSU stimulated BMDMs. (C) NAC reduced XO activity in MSU stimulated BMDMs. (D) NAC reduced IL-1β secretion by MSU stimulated BMDMs, and OKA co-treatment attenuated that effect. (E) Nrf2 expression in BMDMs was enhanced with fingolimod and febuxostat treatment. (F) Representative confocal immunofluorescence images showing enhanced Nrf2 staining in MSU crystal stimulated murine J774 macrophages treated with fingolimod or febuxostat (as shown by arrows). (G) Fingolimod and febuxostat treatments enhanced Nrf2 cellular levels in MSU crystal stimulated J774 macrophages.

Article Snippet: Probing for Nrf2 was performed using anti-Nrf2 rabbit monoclonal antibody (1:1,000; Cell Signaling) overnight at 4°C.

Techniques: Expressing, Activation Assay, Inhibition, Derivative Assay, Incubation, Immunofluorescence, Fluorescence, Control, Generated, Activity Assay, Staining

Journal: Nutrients

Article Title: Oat Peptides Alleviate Dextran Sulfate Sodium Salt-Induced Colitis by Maintaining the Intestinal Barrier and Modulating the Keap1-Nrf2 Axis

doi: 10.3390/nu15245055

Figure Lengend Snippet: OPs regulate the Keap1-Nrf2 signaling axis. ( A ) CCK-8 assay of the effect of OPs on cell proliferation; ( B – D ) Western blotting assay results for Keap1 and Nrf2; ( E , F ) mRNA expression of NQO1 and HO-1; ( G ) Western blotting assay results for HO-1; ( H , I ) immunofluorescence assay results for Keap1 and Nrf2 in Caco-2 cells after OPs treatment. Data are presented as mean ± SD ( n = 3–8 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used in this study: anti-Keap1 antibody (10503-2-AP, Proteintech, Chicago, IL, USA); anti-Nrf2 antibody (16396-1-AP, Proteintech, Chicago, IL, USA); anti-Muc2 antibody (DF8390, Affinity Biosciences, Cincinnati, OH, USA); anti-claudin 1 antibody (AF0127, Affinity Biosciences, Cincinnati, OH, USA); anti-ZO-1 antibody (AF5145, Affinity Biosciences, Cincinnati, OH, USA); anti-occludin antibody (DF7504, Affinity Biosciences, Cincinnati, OH, USA); anti-Alkaline phosphatase antibody (DF6225, Affinity Biosciences, Cincinnati, OH, USA); anti-β-actin antibody (4970, Cell Signaling Technology, Danvers, MA, USA); anti-GaPdh antibody (2118, Cell Signaling Technology, Danvers, MA, USA); anti-HO-1 antibody (A1346, ABclonal, Wuhan, China); anti-rabbit IgG antibody (7074, Cell Signaling Technology, Danvers, MA, USA).

Techniques: CCK-8 Assay, Western Blot, Expressing, Immunofluorescence

Journal: British journal of pharmacology

Article Title: Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy.

doi: 10.1111/bph.15149

Figure Lengend Snippet: FIGURE 4 Metformin promoted mitophagy through activation of AMPK-NRF2. (a) NRF2, NQO1, and HO1 expression levels in whole cell lysates from IEC-6 cells 24 h after 15-Gy radiation. (b) NRF2 expression levels in nuclei from IEC-6 cells 24 h after 15-Gy radiation. (c) NRF2, KEAP1, AMPK, and p-AMPK expression levels in whole cell lysates from IEC-6 cells 24 h after 15-Gy radiation. Dorsomorphin (CC) was used as an AMPK inhibitor. (d) NRF2, NQO1, HO1, Pink1 and P62 expression levels in whole cell lysates from IEC-6 cells with the indicated treatments 24 h after 15-Gy radiation. Dorsomorphin (CC) was used as a NRF2 inhibitor. (e) Intracellular distribution of LC3 and mitochondria in IEC-6 cells 24 h after 15-Gy radiation examined through confocal microscopy

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 62 (#4499S), anti-rabbit AMPKα (#5831S), anti-rabbit PhosphoAMPKα (#2535S) and anti-rabbit Caspase 3 (#9662S) were from Cell Signaling Technologies; anti-mouse (ab56416), anti-mouse Parkin (ab56416), anti-mouse Heme Oxygenase 1(ab13248), anti-rabbit NQO1(ab34173) and anti-65 rabbit Tomm20 (ab186735) were from Abcam; anti-rabbit NRF2 (#16396-1-AP ) Pink1 (#23274-1-AP ) Parkin (#14060-1-AP ) were from Proteintech.

Techniques: Activation Assay, Expressing, Confocal Microscopy

Journal: British journal of pharmacology

Article Title: Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy.

doi: 10.1111/bph.15149

Figure Lengend Snippet: FIGURE 5 Activation of NRF2 is essential for radioprotection of metformin. (a) Relative value of the mitochondria-derived ROS and apoptosis rates (b) were detected in IEC-6 cells 24 h after 15-Gy radiation, both compared with PBS group. (c) PARP, caspase 3 and cleaved caspase 3 expression levels from IEC-6 cells 24 h after 15-Gy radiation. (d) Images (top) of stained by NRF2 immunofluorescence in mice crypts. Red, NRF2; blue, DAPI. Scale bar: 20 μm. Corresponding NRF2 expression levels (bottom) in crypts from mice 24 h after ML385 treatment. (e) Kaplan–Meier survival analysis of mice after 12-Gy whole abdominal radiation (WAI ; n = 10). P < 0.05 for Met + IR versus PBS + IR. (f) Body weight of mice over time after 12-Gy WAI. (g) HE-stained sections of mice intestine after 12-Gy WAI. Scale bars: 500 μm. (h) Average number of surviving crypts per section at the indicated time after 12-Gy WAI. (i) Villi height at the indicated time after WAI. (j) TUNEL+ crypt cells at 4 h after WAI. For subparts (a), (b), (h), (j) and (i), n = 5 (*P < 0.05)

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 62 (#4499S), anti-rabbit AMPKα (#5831S), anti-rabbit PhosphoAMPKα (#2535S) and anti-rabbit Caspase 3 (#9662S) were from Cell Signaling Technologies; anti-mouse (ab56416), anti-mouse Parkin (ab56416), anti-mouse Heme Oxygenase 1(ab13248), anti-rabbit NQO1(ab34173) and anti-65 rabbit Tomm20 (ab186735) were from Abcam; anti-rabbit NRF2 (#16396-1-AP ) Pink1 (#23274-1-AP ) Parkin (#14060-1-AP ) were from Proteintech.

Techniques: Activation Assay, Derivative Assay, Expressing, Staining, Immunofluorescence, TUNEL Assay

Journal: British journal of pharmacology

Article Title: Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy.

doi: 10.1111/bph.15149

Figure Lengend Snippet: FIGURE 8 Proposed model of metformin as radiotherapy adjuvant for P53 mutation colorectal tumours via optimising autophagy. In normal intestinal epithelial cells, metformin promotes AMPK-dependent autophagic degradation of Keap1, then Nrf2 moves to the nucleus and reduces mtROS by transcriptionally activating mitophagy, thereby protecting radiation-induced intestinal injuries. However, in P53 mutation colorectal tumours, protective autophagy is inhibited by mutant p53 protein, resulting in radiosensitive effects

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 62 (#4499S), anti-rabbit AMPKα (#5831S), anti-rabbit PhosphoAMPKα (#2535S) and anti-rabbit Caspase 3 (#9662S) were from Cell Signaling Technologies; anti-mouse (ab56416), anti-mouse Parkin (ab56416), anti-mouse Heme Oxygenase 1(ab13248), anti-rabbit NQO1(ab34173) and anti-65 rabbit Tomm20 (ab186735) were from Abcam; anti-rabbit NRF2 (#16396-1-AP ) Pink1 (#23274-1-AP ) Parkin (#14060-1-AP ) were from Proteintech.

Techniques: Adjuvant, Mutagenesis

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sitagliptin Alleviates Radiation-Induced Intestinal Injury by Activating NRF2-Antioxidant Axis, Mitigating NLRP3 Inf--lammasome Activation, and Reversing Gut Microbiota Disorder

doi: 10.1155/2022/2586305

Figure Lengend Snippet: Sitagliptin upregulates the expression and function of NRF2 in intestinal epithelial cells. (a) mRNA levels of Nrf2, HO-1, and NQO1 in small intestinal tissues. (b) Immunofluorescence of NRF2, HO-1, and NQO1 in small intestinal tissues. Scale bars, 100 μ m. (c) Immunofluorescence of NRF2, HO-1, and NQO1 in HIEC-6 cells. Scale bars, 10 μ m. (d) Western blot analysis of NRF2, HO-1, and NQO1 in HIEC-6 cells. (e–g) Quantitative analysis of NRF2, HO-1, and NQO1 in HIEC-6 cells.

Article Snippet: The small intestines were processed as for IHC, slides were blocked with 10% normal donkey serum and incubated with the primary antibodies anti-NRF2 antibody (1 : 200, CST, #12721), anti- γ -H2AX antibody (1 : 400, CST, #9718), and anti-C-caspase3 (1 : 200, CST, #9664) overnight at 4°C, followed by fluorescent secondary antibody at 37°C for 1 h. Finally, the slides were stained with DAPI (Servicebio, China) and photographed using a fluorescence microscope (Leica, Germany).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response

doi: 10.1038/s12276-021-00596-w

Figure Lengend Snippet: a Decreased gene expression of Nrf2 and Nrf2 antioxidants in the curcumin-treated Optn −/− vs. Optn +/+ osteoclast precursors. Cells were treated with curcumin (1, 5 and 20 µM) for 6 h. Gene expression of the NRF2-mediated antioxidant genes Hmox1 , Gclc , Gclm , and Nqo1 was determined by RT-qPCR with all results were normalized with β-actin. b Decreased protein expression of NRF2 and regulated antioxidants in the curcumin-treated Optn +/+ and Optn −/− preosteoclasts. The cells were treated with curcumin (1, 5 and 20 µM) for 6 h, whole proteins were extracted, and the OPTN, NRF2, HMOX1, and NQO1 levels were measured. c Quantification of WB bands for the protein expression of Nrf2-mediated antioxidants in the curcumin-treated Optn +/+ and Optn −/− preosteoclasts. d Decreased protein levels of NRF2 and key osteoclastogenic markers during osteoclast differentiation in the Optn −/− cells. Primary osteoclast precursors were treated with RANKL (10 ng/ml) and M-CSF (30 ng/mL) for 6 days. At the indicated time points (2, 4, and 6 d), whole protein extracts of the Optn +/+ and Optn −/− cells were extracted and analyzed by Western blotting. e Quantification of WB bands for the protein levels of NRF2 and key osteoclastogenic markers during osteoclast differentiation. CONT, control (growth media only); n = 3 experiments. Data are presented as the mean ± SEM. (a): * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Optn +/+ within each dosage group; † p < 0.05, †† p < 0.01 compared to the untreated control of either the Optn +/+ or Optn −/− group; ( c ): * p < 0.05 compared to the untreated control of either the Optn +/+ or Optn −/− group; e : * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Optn +/+ within each time point group; † p < 0.05, †† p < 0.01 compared to the untreated control of either the Optn +/+ or Optn −/− group.

Article Snippet: After the cells were blocked with horse serum for 24 h, they were incubated with anti-rabbit OPTN (Abcam, ab23666, 1:150) and anti-rat NRF2 (Cell Signaling, #14596, 1:150) primary antibodies; anti-rat NRF2 (Cell Signaling, #14596, 1:150) and anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) primary antibodies; anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) and anti-rabbit SQSTM1/p62 (Abcam, ab240635, 1:150) primary antibodies overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response

doi: 10.1038/s12276-021-00596-w

Figure Lengend Snippet: a Immunoblot analysis of whole-cell lysates and CoIP assays using HEK293T cells. Flag immunoprecipitates were isolated from HEK293T cells transfected with His-OPTN or Flag-Nrf2 plasmids for 1 d. The IP bands showed that Flag-tagged NRF2 can pull down His-tagged OPTN, indicating that NRF2 and OPTN can bind with each other in vitro. β-Actin expression served as a loading control. b Intracellular localization of OPTN and NRF2 in preosteoclasts. The Optn −/− and Optn +/+ preosteoclasts were treated with M-CSF (10 ng/mL) and RANKL (30 ng/mL) for 6, 12, 24, and 72 h. RANKL + 5 μM curcumin treatment for 72 h was used as a positive control. After fixation, the cells were processed with immunofluorescence using antibodies against OPTN (green), NRF2 (red) and nuclei (blue). In merged images, colocalization of OPTN and NRF2 was observed mostly in perinuclear granular structures (yellow). Scale bar = 10 μm.

Article Snippet: After the cells were blocked with horse serum for 24 h, they were incubated with anti-rabbit OPTN (Abcam, ab23666, 1:150) and anti-rat NRF2 (Cell Signaling, #14596, 1:150) primary antibodies; anti-rat NRF2 (Cell Signaling, #14596, 1:150) and anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) primary antibodies; anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) and anti-rabbit SQSTM1/p62 (Abcam, ab240635, 1:150) primary antibodies overnight at 4°C.

Techniques: Western Blot, Isolation, Transfection, In Vitro, Expressing, Positive Control, Immunofluorescence

Journal: Experimental & Molecular Medicine

Article Title: Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response

doi: 10.1038/s12276-021-00596-w

Figure Lengend Snippet: RANKL treatment generates ROS secondary messengers that activate NFATc1 and other genes involved in osteoclastogenesis. ROS homeostasis, including ROS signaling, is maintained by antioxidants, which are in turn transcriptionally regulated by NRF2 and modulated by OPTN.

Article Snippet: After the cells were blocked with horse serum for 24 h, they were incubated with anti-rabbit OPTN (Abcam, ab23666, 1:150) and anti-rat NRF2 (Cell Signaling, #14596, 1:150) primary antibodies; anti-rat NRF2 (Cell Signaling, #14596, 1:150) and anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) primary antibodies; anti-mouse KEAP1 (Santa Cruz Biotechnology, #sc-514914, 1:50) and anti-rabbit SQSTM1/p62 (Abcam, ab240635, 1:150) primary antibodies overnight at 4°C.

Techniques: