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: 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: PLoS ONE

Article Title: Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

doi: 10.1371/journal.pone.0034405

Figure Lengend Snippet: ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.

Article Snippet: DUOX 1 and 2 proteins were detected with a rabbit polyclonal antibody raised against the peptide sequence ETELTPQRLQC located inside the first intracellular loop of human DUOX1; the specificity of the antibodies was tested using human primary thyroid cells as positive control and pre-immune serum as negative control (not shown); goat anti gp91 phox (NOX2) and rabbit p22 phox or SOD1 polyclonal antibodies were purchased by Santa Cruz Biotechnology (USA); The filters were also probed with an anti α-tubulin antibody (Sigma, USA).

Techniques: Transfection, Electroporation, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

doi: 10.1371/journal.pone.0034405

Figure Lengend Snippet: ( A ) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in . ROS levels were measured fluorimetrically at the time intervals indicated. Ca ++ -independent ROS were measured in presence of BAPTA-AM. Ca ++ -dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. ( B ) Cells were transfected by electroporation with siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H 2 O 2 levels as described in . The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.

Article Snippet: DUOX 1 and 2 proteins were detected with a rabbit polyclonal antibody raised against the peptide sequence ETELTPQRLQC located inside the first intracellular loop of human DUOX1; the specificity of the antibodies was tested using human primary thyroid cells as positive control and pre-immune serum as negative control (not shown); goat anti gp91 phox (NOX2) and rabbit p22 phox or SOD1 polyclonal antibodies were purchased by Santa Cruz Biotechnology (USA); The filters were also probed with an anti α-tubulin antibody (Sigma, USA).

Techniques: Incubation, Derivative Assay, Transfection, Electroporation

Journal: PLoS ONE

Article Title: Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse

doi: 10.1371/journal.pone.0015354

Figure Lengend Snippet: A. Representative western blots of all NADPH oxidase subunits from TA muscles of 18–19 day old WT and mdx mice. B. Pooled results from densitometry for the subunits showing mdx values normalized to WT (represented by the blue dotted line). The proteins gp91 phox , p67 phox and rac1 were all significantly increased in mdx muscle compared to WT, whereas p22 phox and p47 phox were not different (n = 4 samples for each group).

Article Snippet: Membranes were probed using mouse antibodies against rac1 (1∶500) (Cytoskeleton, CO), gp91 phox (1∶1000) and p67 phox (1∶500) (BD Biosciences, San Jose, CA), and rabbit antibodies against p22 phox (1∶500) and p47 phox (1∶500) (Santa Cruz, CA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse

doi: 10.1371/journal.pone.0015354

Figure Lengend Snippet: Isolated mdx muscle fibers from the FDB muscle were immunostained using antibodies against the various NADPH oxidase subunits. Nuclei are stained by DAPI (blue). In the bottom right panel, p22 phox (green) was co-immunostained with caveolin-3 (red) to demonstrate sarcolemmal localization (yellow).

Article Snippet: Membranes were probed using mouse antibodies against rac1 (1∶500) (Cytoskeleton, CO), gp91 phox (1∶1000) and p67 phox (1∶500) (BD Biosciences, San Jose, CA), and rabbit antibodies against p22 phox (1∶500) and p47 phox (1∶500) (Santa Cruz, CA).

Techniques: Isolation, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia

doi: 10.1155/2018/3273654

Figure Lengend Snippet: Expression of NADPH oxidase subunits: (a) gp91 phox , (b) p22 phox , (c) NOX4, (d) p47phox, and (e) p67 phox . Group I ( n = 6) was sham-operated animals, which underwent the same surgical procedure without vessel occlusion (control), group II ( n = 6) was subjected to ischemia/reperfusion (I/R), and group III ( n = 5) was treated with vitamin D3 and then subjected to I/R (VitD + I/R). All experiments were performed in synaptosomal fraction in different brain structures. Results are presented as mean ± SE. ∗ p < 0.05 versus control, ∗∗ p < 0.01 versus control, § p < 0.05 versus I/R, and §§ p < 0.01 versus I/R.

Article Snippet: The following primary antibodies were used in this study: mouse polyclonal NeuN (1 : 1000, Millipore, Germany), mouse polyclonal 91- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p22- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p47- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p67- phox (1 : 500, Santa Cruz, CA), rabbit monoclonal NOX4 (1 : 5000, Abcam, UK), rabbit polyclonal VDR (1 : 1000, Abcam, UK), goat polyclonal anti-Iba1 (1 : 250, Abcam, UK), and rabbit polyclonal anti-MMP9 (1 : 1000, Sigma-Aldrich, Germany).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia

doi: 10.1155/2018/3273654

Figure Lengend Snippet: Immunohistochemical staining of gp91 phox and p22 phox in gerbil brain slices. Magnification of images for (a) from A–F is 200×, for (b) from A–C is 40×, and for (b) from D–I is 100×.

Article Snippet: The following primary antibodies were used in this study: mouse polyclonal NeuN (1 : 1000, Millipore, Germany), mouse polyclonal 91- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p22- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p47- phox (1 : 500, Santa Cruz, CA), rabbit polyclonal p67- phox (1 : 500, Santa Cruz, CA), rabbit monoclonal NOX4 (1 : 5000, Abcam, UK), rabbit polyclonal VDR (1 : 1000, Abcam, UK), goat polyclonal anti-Iba1 (1 : 250, Abcam, UK), and rabbit polyclonal anti-MMP9 (1 : 1000, Sigma-Aldrich, Germany).

Techniques: Immunohistochemical staining, Staining