Journal: Redox Biology

Article Title: SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

doi: 10.1016/j.redox.2021.102041

Figure Lengend Snippet: Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression ( A , left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi ( A , right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins ( B ), and GCLC protein ( C ) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of . Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used were: Nrf2 Polyclonal Antibody, FITC Conjugated (bs-1074R-FITC, Bioss Antibodies, 1:50); NQO1 (A180, Mouse mAb #3187, CST, 1:50); GST-pi (610,718, mouse mAb, BD Biosciences, 1:500); PE anti-human IL-6 Antibody (BioLegend, 1:1000); PE anti-human IL-10 Antibody (BioLegend, 1:1000).

Techniques: Infection, Western Blot, Expressing, Fluorescence

Journal: Frontiers in Medicine

Article Title: Restored retinal physiology after administration of niacin with citicoline in a mouse model of hypertensive glaucoma

doi: 10.3389/fmed.2023.1230941

Figure Lengend Snippet: Western blot antibodies.

Article Snippet: Rabbit polyclonal anti-Nrf-2 , 1:1000 , Abcam (Cambridge, UK) , ab92946.

Techniques: Western Blot

Journal: Frontiers in Medicine

Article Title: Restored retinal physiology after administration of niacin with citicoline in a mouse model of hypertensive glaucoma

doi: 10.3389/fmed.2023.1230941

Figure Lengend Snippet: Effect of individual or combined administration of niacin and citicoline on MCE-induced oxidative stress. (A) Representative Western blots and densitometric analysis of nuclear factor erythroid 2-related factor-2 (Nrf-2) and (B) heme oxygenase-1 (HO-1) levels in control and MCE mice either untreated or treated with individual niacin and citicoline or their combination. Data are expressed as mean ± SEM ( n = 6 retinas for group). * p < 0.0001 vs. control; § p < 0.01, §§ p < 0.001, and §§§ p < 0.0001 vs. MCE (two-way ANOVA followed by Tukey’s multiple comparison post-hoc test).

Article Snippet: Rabbit polyclonal anti-Nrf-2 , 1:1000 , Abcam (Cambridge, UK) , ab92946.

Techniques: Western Blot, Comparison

Journal: Aging

Article Title: Pharmacological recapitulation of the lean phenotype induced by the lifespan-extending sulfur amino acid-restricted diet.

doi: 10.18632/aging.206237

Figure Lengend Snippet: Figure 3. The SAAR diet and BSO exert tissue-specific effects on Nrf2 and Phgdh. The SAAR diet increased Nrf2 (A) and Phgdh (B) protein expressions in the liver, which ultimately resulted in higher serine concentrations (C). Unlike the SAAR diet, BSO did not increase Nrf2 and Phgdh in the liver but increased both in the kidneys (D, E). Regardless of the changes in Nrf2 and Phgdh, BSO increased serine concentrations in livers and kidneys (C–F). NAC reversed SAAR-induced changes in Nrf2, Phgdh, and serine (A–F). Note: Sample size = 5-6. Statistical methods are similar to those in Figure 2.

Article Snippet: Host species Antibody dilutions Primary Antibodies Nrf2 Cell Signaling Technology 20733 Rabbit 1:1000 in 5% BSAa Phgdh Cell Signaling Technology 13428 Rabbit 1:1000 in 5% milka β-Actin Sigma A5441 Mouse 1:15000 in 5% milka Vinculin Proteintech 66305-1-Ig Mouse 1:10000 in 5% milka Secondary Antibodies Anti-Rabbit-HRP Cell Signaling Technology 7074 Goat 1:4000 in 5% milk, 1hb Anti-Mouse-HRP Bio-Rad 170-6516 Goat 1:20000 in 5% milk, 30 minb aAll incubations were performed overnight at 4° C. bAll incubations were performed at room temperature. www.aging-us.com 22 AGING Supplementary Table 3.

Techniques:

Journal: Journal of Microbiology and Biotechnology

Article Title: Fisetin Protects C2C12 Mouse Myoblasts from Oxidative Stress-Induced Cytotoxicity through Regulation of the Nrf2/HO-1 Signaling

doi: 10.4014/jmb.2212.12042

Figure Lengend Snippet: As an activator of Nrf2 and a scavenger of ROS, fisetin protected cells from apoptosis by blocking H 2 O 2 -induced DNA and mitochondrial damage and cell cycle arrest.

Article Snippet: Primary antibodies against Nrf2 (Mouse monoclonal, 1:1000, sc-518036), HO-1 (Mouse monoclonal, 1:1000, sc-136960), p21 (Mouse monoclonal, 1:1000, sc-271610), cyclin A (Mouse monoclonal, 1:1000, sc-271645), Bax (Mouse monoclonal, 1:1000, sc-7480), Bcl-2 (Mouse monoclonal, 1:1000, sc-509), caspase-3 (Mouse monoclonal, 1:1000, sc-56052), poly(ADP-ribose) polymerase (PARP, mouse monoclonal, 1:1000, sc-8007), cytochrome c (Mouse monoclonal, 1:500, sc-13560), actin (Mouse monoclonal, 1:1000, sc-47778) and secondary antibodies were from Santa Cruz Biotechnology Inc. (USA).

Techniques: Blocking Assay

Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs activates the Nrf2 pathway and induces Nrf2-mediated gene expression. (A and B) (Left) RAW 264.7 cells stably expressing the vector, NSs-WT, or NSs-A46 were harvested, and WCEs (A) and the cytoplasmic/nuclear fractions (B) were analyzed by immunoblotting for the indicated proteins. (Right) The relative levels of Nrf2 in RAW 264.7 cells compared to the levels of β-actin for WCEs (A) and p84 for the nucleus (B) were calculated. (C and D) The mRNA levels of the Nrf2 target genes (Hmox1 and Nqo1) (C) and Nrf2 (D) in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 were measured by qRT-PCR. (E) The Nqo1 mRNA level in RAW 264.7 cells treated with dimethyl sulfoxide (DMSO) and an Nrf2 inhibitor (INH; 10 μM) for 24 h was measured by qRT-PCR.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Gene Expression, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway

doi: 10.1128/JVI.01684-19

Figure Lengend Snippet: NSs induces CD36 expression via the Nrf2 pathway and increases lipid uptake. (A) The level of Cd36 mRNA in RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by qRT-PCR. (B) CD36 surface expression by RAW 264.7 cells expressing the vector, NSs-WT, or NSs-A46 was measured by flow cytometry. The surface expression of FITC fluorescence on cells was determined using FlowJo software (left), and the relative expression levels are provided (right). FACS, fluorescence-activated cell sorting. (C) The level of Cd36 mRNA in RAW 264.7 cells treated with DMSO or an Nrf2 inhibitor (INH, 10 μM) for 24 h was measured by qRT-PCR. (D) RAW 264.7 cells were treated with rabbit IgG-FITC complexed with latex beads at a final dilution of 1:500. (Right) The cells were washed/harvested, and flow cytometry was applied to measure the internalized rabbit IgG-FITC-complexed latex beads. (Left) The percentage of cells that phagocytosed the beads was determined using FlowJo software. (E) The organic phase of RAW 264.7 cells was extracted to measure the amount of free or total (free and esterified) cholesterol. A colorimetric assay was used to measure intracellular cholesterol levels at an absorbance of 570 nm, based on cholesterol standards. (F) RAW 264.7 cells were plated and treated with fluorescently tagged cholesterol at 20 μg/ml for 48 h. The cells were washed, fixed with paraformaldehyde, and applied to a microplate reader with filter sets designed for FITC and GFP. The amount of cholesterol taken up was described at a relative level.

Article Snippet: The primary antibodies included TRIM21 (clone D1O1D; Cell Signaling), Nrf2 (clone D1Z9C; Cell Signaling), p62 (Cell Signaling), Keap1 (clone D6B12; Cell Signaling), GFP (clone B-2; Santa Cruz), GST (clone B-14; Santa Cruz), β-actin (clone C4; Santa Cruz), Flag (for rabbit Flag, clone F7425 [Sigma]; for mouse Flag, clone F1804 [Sigma]), HA (for rabbit HA, clone PRB-101P [Covance]; for mouse HA, clone 16B12 [BioLegend]), V5 (for rabbit V5, clone A190 [Bethyl]; for mouse V5, Invitrogen), and Myc (for rabbit Myc, clone Poly9063 [BioLegend]; for mouse Myc, clone 9E10 [BioLegend]).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Software, FACS, Colorimetric Assay