Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: VEO-IBD NOX1 mutations abolish catalytic activity and prevent peroxynitrite formation. (A) Superoxide generation of CHO-NOXO1-NOXA1-p22 phox cells (control) or cells co-expressing NOX1 WT, NOX1 N122H or NOX1 T497A in the presence or absence of PMA (in RLU and as % of WT). (B) Immunoblot of cell lines in (A). (C) Detection of peroxynitrite with FIBA in the absence or presence of 10 μM l -NAME (in RFU). Cell lines in (A) co-expressing iNOS as indicated were stimulated with PMA. (D) Nitrate quantification in supernatants of indicated cell lines after PMA stimulation (1 h). (E) Immunoblot of cell lines in (C). A two-way ANOVA with multiple comparisons test was performed (A) or a Kruskal-Wallis test with multiple comparisons (D). (non-significant (NS) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Article Snippet: Primary antibodies used were NOX1 [ ]; NOX2 (53/gp91 phox , BD); V5-tag (V5-10), Vinculin (hVIN-1), β-Tubulin (ONS.1A6), F-Actin (polyclonal) (all Sigma-Aldrich); HA-tag (16B12, Covance); p22 phox (FL-195, Santa Cruz); p47 phox and p67 phox [ ].

Techniques: Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: Conserved asparagine in the HxxxHxxN motif is essential for the catalytic activity of NOX/DUOX. (A) NOX1-5 and DUOX1-2 sequence alignments with asparagine (blue) and histidines (yellow), and with threonine (blue) and GRP sequence (yellow). (B) CHO-NOXO1-NOXA1-p22 phox cell lines were transfected with NOX1 WT and mutants, followed by PMA stimulation. (C) COS7-p22 phox cells were transfected with p47 phox , p67 phox , and NOX2 WT or mutants, followed by PMA stimulation. (D) COS7-p22 phox cells were transfected with NOX4 WT or mutants, constitutive H 2 O 2 generation was measured. (E) H661-DUOXA2 cells were transfected with DUOX2 or mutants, PMA/thapsigargin stimulated H 2 O 2 generation was measured. (F) Percentage of NOX4 or DUOX2 positive cells by cell surface staining, transfections as above. A one-way ANOVA with multiple comparisons (NOX1,2) or Kruskal-Wallis test with multiple comparisons (NOX4, DUOX2) was performed (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used were NOX1 [ ]; NOX2 (53/gp91 phox , BD); V5-tag (V5-10), Vinculin (hVIN-1), β-Tubulin (ONS.1A6), F-Actin (polyclonal) (all Sigma-Aldrich); HA-tag (16B12, Covance); p22 phox (FL-195, Santa Cruz); p47 phox and p67 phox [ ].

Techniques: Activity Assay, Sequencing, Transfection, Staining

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: NOX1 modelling reveals structural features involved in the oxygen reduction center. (A) Cartoon representation of a NOX1/p22 phox heterodimer model inside view (left) or eclipsed (p22 phox behind, center). On the right, eclipsed view in surface mode revealing lateral access to the distal heme (zoom), NOX1 (teal blue), p22 phox (orange). (B) Zoom, on a vertical section within NOX1, on the distal heme region in NOX1 with cavities highlighted in surface mode. An additional internal pocket at the distal heme site is revealed. The side chains of the heme-chelating histidines as well as N122 and H119, delimiting the internal pocket, are represented by sticks. The vertical section within NOX1 allows to see only TM3, TM4 and partly TM5, while the others have been eliminated from front for clarity. (C) Zoom in NOX1 structure of the N122 environment at the interface between TM3 and TM2 observed from the outside (left) or the inside of NOX1 (right). (D) Modelling the structural impact of mutations at position N122. Modification performed by the use of the mutagenesis function within Pymol software. From left to right, the N122H, N122Q, N122L and N122T mutations. For the first two, several possible rotamers have been represented simultaneously for the corresponding mutated side chain. In the case of N122L and N122T modeled mutations another rotamer of the facing T49 side chain has been considered as possible adaptation to the mutation of N122. In (C) and (D), dotted yellow line represented H-bond and dotted green line possible hydrophobic interaction. (E) CHO-NOXO1-NOXA1-p22 phox cells were transfected with NOX1 WT and mutants, followed by PMA stimulation. Immunoblots indicate equal protein expression. One-way ANOVA with repeated measures (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used were NOX1 [ ]; NOX2 (53/gp91 phox , BD); V5-tag (V5-10), Vinculin (hVIN-1), β-Tubulin (ONS.1A6), F-Actin (polyclonal) (all Sigma-Aldrich); HA-tag (16B12, Covance); p22 phox (FL-195, Santa Cruz); p47 phox and p67 phox [ ].

Techniques: Modification, Mutagenesis, Software, Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: A disease-associated gene desert orchestrates macrophage inflammatory responses via ETS2

doi: 10.1101/2023.05.05.539522

Figure Lengend Snippet: a. Schematic of experiment for disrupting ETS2 in primary monocytes and differentiating monocyte-derived macrophages under chronic inflammatory conditions. b. Macrophage cytokine secretion following ETS2 disruption. Heatmap shows log2 fold-change of cytokine concentrations in the supernatants of ETS2 -edited macrophages relative to unedited macrophages transfected with a non-targeting control gRNA-containing RNP (NTC). n=9, Wilcoxon matched-pairs test, two-tailed. c. Histogram depicting phagocytosis of fluorescently-labelled zymosan particles by ETS2 -edited and unedited macrophages (left). Data representative of one of seven donors. Phagocytosis index in ETS2 -edited and unedited macrophages (calculated as product of proportion and mean fluorescence intensity of phagocytosing cells; right). Plot depicts log2 fold-change in phagocytosis index for ETS2 -edited macrophages relative to unedited cells (Wilcoxon signed-rank test, two-tailed; data represent mean±SEM). d. Production of ROS by ETS2 -edited and unedited inflammatory macrophages (measured in relative light units; left). Data representative of one of six donors. Western blot for gp91 phox , p22 phox , and EROS expression in ETS2 -edited and unedited macrophages (right). Data representative of one of three donors. e. Differentially-expressed genes in ETS2 -edited versus unedited inflammatory macrophages (limma with voom transformation, n=8). f. Gene set enrichment analysis (fGSEA) of differentially-expressed genes between ETS2 -edited and unedited inflammatory macrophages. Results of selected Gene Ontology Biological Pathways shown. Dot size represents P -value and colour denotes normalised enrichment score (NES). g. Enrichment of differentially-expressed genes following deletion of the disease-associated chr21q22 locus (upregulated genes, top; downregulated genes, bottom) in ETS2 -edited versus unedited macrophages. * P < 0.05, ** P < 0.01.

Article Snippet: Western blotting was performed as described previously using the following primary antibodies: rabbit anti-gp91 phox , rabbit anti-p22 phox (both Santa Cruz), rabbit anti-C17ORF62/EROS (Atlas), rabbit anti-actin (Abcam).

Techniques: Derivative Assay, Transfection, Two Tailed Test, Fluorescence, Western Blot, Expressing, Transformation Assay

Journal: Toxics

Article Title: Hypoxia-Induced Kidney Injury in Newborn Rats

doi: 10.3390/toxics11030260

Figure Lengend Snippet: Hypoxic kidney injury involves oxidative stress, apoptosis, and inflammatory signaling. ( A ) Western blot analysis shows the oxidative-stress-related proteins, including P22, P47, NOX2, and NOX4. ( B ) Western blot analysis shows apoptosis-related proteins, including p-P38, Casp-9, and cleaved casp-3. ( C ) Western blot analysis shows the inflammatory-related proteins, including TNF-α and NF-κB. Quantitative analysis was performed for each blot. The values are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, versus the normoxic group. The p -values were estimated via Mann–Whitney U test ( n = 6). N, normoxia; H, hypoxia. P22, nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase subunit p22-phox; P47, NADPH oxidase subunit p47-phox; NOX2, NADPH oxidase 2; NOX4, NADPH oxidase 4; p-P38, phospho-p38 mitogen-activated protein kinase (Thr180/Tyr182); P38, p38 MAP kinase; Casp-9, caspase-9; cleaved Casp 3, cleaved-caspase-3; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

Article Snippet: The membrane was blocked in PBST buffer with 5% nonfat milk and 5% bovine serum albumin before being incubated with anti-hypoxia-inducible factors, alpha subunit (HIF-1α) (20960–1AP, Proteintech), anti-heme oxygenase-1 (HO-1) (ADI SPA-895, Enzo Biochem, Inc., Farmingdale, NY, USA), anti-NGAL (ab63929, Abcam), anti- nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase subunit p22-phox (P22) (sc271968, Santa Cruz Biotechnology, Dallas, TX, USA), NADPH oxidase subunit p47-phox (P47) (sc-17845, Santa Cruz), anti- NADPH oxidase 2 phox (NOX2) (ab129068, Abcam), anti- NADPH oxidase 4 phox (NOX4) (14347–1-Ig, Proteintech), anti-collagen type I (67288–1-Ig, Proteintech), anti-fibroblast growth factor 23 (FGF23) (Ls-C411984, Lifespan Biosciences, Seattle, WA, USA), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) (p-P38) (#9211, Cell signaling, Danvers, MA, USA), p38 MAPK (P38) (9212S, Cell Signaling), anti-caspase-9 (GTX112888, GeneTex, Irvine, CA, USA), anti-cleaved caspase-3 (#9664, Cell Signaling), anti-tumor necrosis factor-α (TNF-α) (PA1079, Boster biological technology, Pleasanton, CA, USA), anti-nuclear factor kappa B (NF-κB) (10745–1-AP, Proteintech), anti-actin (MAB1501, Merck KGaA, Darmstadt, Germany), anti-alpha tubulin (ab7291 Abcam), and anti-GAPDH (60004–1-Ig, Proteintech) antibodies.

Techniques: Western Blot, Standard Deviation, MANN-WHITNEY

Journal: Antioxidants

Article Title: Targeting M2 Macrophages with a Novel NADPH Oxidase Inhibitor

doi: 10.3390/antiox12020440

Figure Lengend Snippet: Confocal imaging of living cells (Left A – F) ( A , B ): monocytes isolated from blood of healthy donors; the red signal is the NOX2 and mitotracker in ( A , B ), respectively, the grey signal corresponds to the mitotracker and CellRox deep red reagent in ( A , B ), respectively; ( C , D ) Monocyte-derived macrophages (with CSF1-without NS1 treatment) using the same markers used for the living monocytes in ( A , B ); ( E , F ) the macrophages were treated with NS1 after their CSF1-induced differentiation from monocytes using the same markers as in ( A , B ); the green signal in ( F ) is the fluorescence of NS1; the ROS levels are quantified by the cell-permeable CellROX deep red reagent shown in light grey, the mitochondria are monitored by the red mitotracker reagent as quantified in ( G ); Co-localization experiments in fixed cells (Right H – L) The images ( H – L ) present co-localization experiments in fixed monocytes or CSF1-treated monocytes differentiated into macrophages . Each antibody is mentioned above each image, the merge, representing the colocalization is shown in yellow. The intensity of the colocalization is quantified in ( M ). We tested colocalization between partners of the activated NOX2 complex in “M2” macrophages formed by membrane bound NOX2 and p22 phox with p67 phox and p47 phox (p40 phox and rac1 are not labeled). It is known that p67 phox and p47 phox are located in the cytoplasm of the resting cells in agreement with the images shown in ( H ); ( I , J ) are p22 phox -p47 phox and p22 phox -p67 phox co-localization signals in macrophages without NS1, respectively; ( K , L ) are p22 phox -p47 phox and p22 phox -p67 phox co-localizations with NS1, respectively. Scale bars are 3 µm. Quantification of the 3D images is shown in ( M ) and was performed with Imaris 7.3 software ( http://www.bitplane.com/imaris , accessed on 6 February 2023).

Article Snippet: Mitotracker (red or far-red) and CellRox deep red were purchased from Thermo-Fischer and Invitrogen (Les Ulys, France), respectively; antibodies against NOX2 (ab80897) and p22 phox (ab75941) were acquired from Abcam; p47 phox (sc7660) and p67 phox (sc7663) were acquired from Santa Cruz; and NS1 was synthesized by Innoverda according to the published synthesis [ ].

Techniques: Imaging, Isolation, Derivative Assay, Fluorescence, Labeling, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Evaluation of Effects of Chinese Prescription Kangen-karyu on Diabetes-Induced Alterations such as Oxidative Stress and Apoptosis in the Liver of Type 2 Diabetic db/db Mice

doi: 10.1155/2012/143489

Figure Lengend Snippet: Nox-4 (a) and p22 phox (b) protein expressions in the liver. m/m : Misty; Veh: vehicle-treated db/db mice; K-100: Kangen-karyu 100 mg/kg body weight-treated db/db mice; K-200: Kangen-karyu 200 mg/kg body weight-treated db/db mice. Bars represent the means ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001 versus vehicle-treated db/db mouse values.

Article Snippet: Rabbit polyclonal antibodies against p22 phox , NF- κ Bp65, nuclear factor erythroid 2-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1), cytochrome c , Bax, and mouse monoclonal antibodies against cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), Bcl-2, and histone were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

doi: 10.1186/1742-2094-8-129

Figure Lengend Snippet: Dopaminergic cells express subunits of the NADPH oxidase complex . (A) mRNA from untreated N27 cells was reverse transcribed and amplified using PCR primers specific to rat NADPH oxidase subunits. Nox1-4 subunits as well as p22 phox , p40 phox , p47 phox , and p67 phox were identified by their mRNA expression. Nox2, p22 phox , and p47 phox were also detected by their protein expression (Western immunoblot, B-D) and cellular localization (immunofluorescence, B-D). Scale bar equals 10 μm; all three micrographs were taken at the same magnification.

Article Snippet: Anti-p47 phox and anti-Nox2 antibodies were from Upstate Biotechnology; anti-p22 phox and anti-p67 phox antibodies were from Santa Cruz Biotechnology; and anti-TH from Pel-Freez Biologicals.

Techniques: Amplification, Expressing, Western Blot, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

doi: 10.1186/1742-2094-8-129

Figure Lengend Snippet: Pharmacological inhibitors of NADPH oxidase and silencing p22 phox using siRNA attenuate MPP+ induced ROS . N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H 2 O 2 levels were measured using caboxy-H 2 -DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22 phox . Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22 phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22 phox siRNA Smartpool and the intracellular H 2 O 2 was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. Data are from 3 independent experiments with n = 6 wells per experiment.

Article Snippet: Anti-p47 phox and anti-Nox2 antibodies were from Upstate Biotechnology; anti-p22 phox and anti-p67 phox antibodies were from Santa Cruz Biotechnology; and anti-TH from Pel-Freez Biologicals.

Techniques: Flow Cytometry, Transfection, Staining, Agarose Gel Electrophoresis, Produced

Journal: PLoS ONE

Article Title: Oxidized LDL Triggers Pro-Oncogenic Signaling in Human Breast Mammary Epithelial Cells Partly via Stimulation of MiR-21

doi: 10.1371/journal.pone.0046973

Figure Lengend Snippet: ( A ) – Western blots for Lipoxygenases-12, lipoxygenase -15-2 and P22 phox and P47 phox subunits of NADPH oxidase (treatment with 20 µg/ml ox-LDL for 2 and 12 hours); ( B ) – corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change); ( C ) – Western blots for SOD1 and SOD2 (similar conditions) and ( D ) – corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change). (*) – Significant difference (p<0.05) compared to control.

Article Snippet: Source of antibodies to LOX-1, CD36, CXLC16, MSR1, lipoxygenase-12 and lipoxygenase-15 was AbCam (Cambridge, MA), antibodies to SOD1 and SOD2 was Enzo Life Sciences (Exeter, UK), antibodies to PI3K, PTEN, and Akt was Cell Signaling Technology (Danvers, MA), and antibodies to NADPH oxidase (subtypes P47 phox and P22 phox ) was Santa Cruz (Santa Cruz, CA).

Techniques: Western Blot