Journal: Biomolecules & Therapeutics

Article Title: Niclosamide Inhibits Aortic Valve Interstitial Cell Calcification by Interfering with the GSK-3β/β-Catenin Signaling Pathway

doi: 10.4062/biomolther.2022.146

Figure Lengend Snippet: Niclosamide inhibits PCM induced ROS generation in VICs. (A) Representative fluorescence microscopy images of DCFDA and DHE in live-adherent VICs with corresponding DAPI staining. (B) Relative level of ROS in VICs determined by microplate assay and were calculated as a percentage of CM. Results are expressed as mean ± SEM (n=3). *** p <0.001 vs. CM; # p <0.05; ## p <0.01 vs. PCM. (C) Relative level of NADPH oxidase in VICs was determined by lucigenin chemiluminiscence assay and chemiluminiscence intensities were calculated as a percentage of CM. Results are expressed as mean ± SEM (n=3). ** p <0.01 vs. CM; # p <0.05; ## p <0.01; ### p <0.001 vs. PCM. (D, E) The gene expression of NOX2 and its catalytic subunit p22 phox were determined by RT-qPCR. Results are expressed as mean ± SEM (n=4). ** p <0.01; *** p <0.001 vs. CM; # p <0.05; ## p <0.01; ### p <0.001 vs. PCM. (F, G) The protein expression of Nox2 and p22 phox were determined by western blotting and quantification analysis. Results are expressed as mean ± SEM (n=4). * p <0.05; ** p <0.01; vs. CM; # p <0.05; ## p <0.01 vs. PCM.

Article Snippet: The following are the primary antibodies used in this study: Runx2 (1:2,000, LsBio, Seattle, WA, USA), OPN (1:2,000, Abcam, Cambridge, UK), Nox2 (1:2,000, Proteintech, Rosemont, IL, USA), p22 phox (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (1:2,000, Cell Signaling Technology, Beverly, MA, USA), p-AKT (1:2,000, Cell Signaling Technology), t-AKT (1:2,000, Cell Signaling Technology), p-ERK (1:2,000, Cell Signaling Technology), t-ERK (1:2,000, Cell Signaling Technology) and secondary antibodies [horseradish peroxidase (HRP) - conjugated anti-mouse or anti-rabbit immunoglobulin G (1:20,000; Cell Signaling Technology)].

Techniques: Fluorescence, Microscopy, Staining, Expressing, Quantitative RT-PCR, Western Blot

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