Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: Vertical scatter plot of NOX2 and NOX4 expression data from the Tomlins cohort ( A and D respectively) consisting of 18 non-malignant tissues, 13 prostatic intraepithelial neoplasia’s, 30 primary prostate cancer and 19 metastatic cancer tissue samples. NOX2 and NOX4 expression displayed as percentage-change from the Taylor cohort ( B and E respectively) consisting 29 non-malignant and 131 primary-cancer tissue samples. Log2 median-centred ratio of NOX2 and NOX4 from the Grasso cohort ( C and F respectively) consisting 59 cancer 28 normal tissue samples. * P < 0.05, ** P < 0.01 and **** P < 0.0001 for Students t test (C, E and F) or one-way ANOVA (A and D).

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques: Expressing

Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: ( A ) The effect of the VEGFR2 inhibitor Ki8751 (25 mg/kg/day, i.p) administered from Day 10 on prostate tumour growth in mice after 14 days ( n = 8). ( B ) The data and ( C ) representative images showing the growth of prostate tumours over 14 days in WT and NOX2 -/y mice ( n = 8–15). For (A) and (B) the prostate weights include the prostate and any associated tumour plus seminal vesicles. ( D ) Representative images of the density of CD31 + cells in prostate tumours in WT mice at Day 14. Note, the image in the NOX2 -/y represents the CD31+ staining in one of the larger tumours formed in the NOX2 -/y mice group. Horizontal black bar represents the scale of 200mm. The graph in (D) shows the average number of CD31 + cells per field in each group. ( E ) Representative images of the density of VEGFR2 + cells in prostate tumours in WT mice at Day 14. Horizontal black bar represents the scale of 200 mm. The graph in (E) shows the average number of VEGFR2 + cells per field in each group. ( F ) Group data showing effect of apocynin (50 mg/kg/day i.p. and 500 mg/L drinking water) on tumour development at Day 14 when administered in WT mice bearing tumours at Day 10 ( n = 8). Data are mean ± SEM. Students unpaired t test (A, D, F) or one-way ANOVA (B and G). * P < 0.05, ** P < 0.01.

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques: Staining

Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: ( A – B ) Confocal fluorescent images showing co-located NOX2 (red) with endosome markers (green) (A) Rab5 and (B) Rab7 in non-malignant (PNT1A) and malignant (LNCaP) human prostate cancer cells and in LNCaP post-VEGF-A treatment. ( C ) Graphs show the degree of co-location of NOX2 with the endosome markers Rab5 and Rab7 in VEGF-A-treated and untreated PNT1a and LNCaP cells. Data is representative of 6 randomly selected cells and are shown as mean ± SEM. * P < 0.05, ** P < 0.01 for two-way ANOVA.

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques:

Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: ( A ) Confocal immunofluorescent images showing NOX2 (red) with the endosome markers Appl1, Rab5A, EEA1 and Rab7A (green) co-location within HMEC-1 cells after 30 min incubation with either PBS or VEGF-A (30 ng/mL). ( B ) Graph shows the degree of co-localisation (Pearson’s correlation) within the cells. Data are mean ± SEM. ** P < 0.01 for Mann–Whitney unpaired t test.

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques: Incubation, MANN-WHITNEY

Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: ( A ) Confocal fluorescence images of HMEC-1 incubated with OxyBURST green for 5 min before incubating for 30 min with either PBS, VEGF-A (30 ng/mL) or VEGF-A (100 ng/mL). ( B ) Graphs showing the area of fluorescence and mean fluorescence per cell detected in each group ( n = 5). ( C ) Confocal images of time course of endosomal ROS generation in HMEC-1 cells. Cells were incubated with OxyBURST green for 5 min and then either PBS for 30 min (control) or VEGF-A (30 ng/mL) for between 5 and 30 min ( n = 3). ( D ) Confocal images of endosomal ROS generation after 30 min incubation with VEGF-A (30 ng/mL) and 30 min of incubation with either SOD (100 U/mL), apocynin (300 µM) or bafilomycin A (10 nM) followed by 30 min incubation with VEGF-A (30 ng/mL). ( E ) Graphs show total number of ROS-producing cells expressed as percentages of the total number of cells per group plus the mean fluorescence per ROS-producing cell ( n = 3-5). ( F ) Confocal images of endosomal ROS generation after 30 min incubation with VEGF-A (30 ng/mL) and ( G ) their corresponding graphs showing total number of ROS-producing cells expressed as percentages of the total number of cells per group plus the mean fluorescence per ROS-producing cell for each group in wild-type and NOX2 knockout (NOX2 -/y ) mouse lung endothelial cells (MLEC cells; n = 4). ( H ) Extracellular H 2 O 2 production as assessed by Amplex Red fluorescence in the absence or presence of VEGF-A ( n = 5). Data are mean ± SEM. * P < 0.05, ** P < 0.01 for Students unpaired t test (E and G) or one-way ANOVA (B and E).

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques: Fluorescence, Incubation, Knock-Out

Journal: Oncotarget

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

doi: 10.18632/oncotarget.26237

Figure Lengend Snippet: ( A – F ) The proportion of HMEC-1 per well after 24 hr treatment with either PBS or VEGF-A (30 ng/mL) in the absence or presence of either (A) Dynasore (Dyna; 100 µM), (B) pitstop 2 (Pit; 30 µM), (C) bafilomycin A (Baf; 10 nM), (D) catalase (Cat; 1000 U/mL), (E) SOD (100 U/mL) or (F) apocynin (Apo; 300 µM). ( G ) Graph shows the effects of VEGF-A (10 and 30 ng/mL) on WT and NOX2 -/- mouse lung endothelial cell proliferation after 24 hr expressed as percentages of the PBS control. Data are mean ± SEM for n = 5-7 experiments. * P < 0.05, ** P < 0.01, for one-way ANOVA.

Article Snippet: Ltd, Australia), rabbit mAb VEGF receptor 2 antibody (1:1000; Cell Signaling, Australia) and mouse anti-gp91phox antibody (1:1000; BD Biosciences, Australia) overnight at 4° C. Cells were then given 4 × 10 min washes in 0.01 M PBS before being incubated with secondary antibodies (1:1000 for each of Alexa fluor 647 goat anti-rabbit and 488 goat anti-mouse, both Life Technologies) for 2 hr at room temperature.

Techniques:

Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 3. Comparison of gp91phox, Poldip2 and p22phox expression in monocytes, neutrophils and macrophages isolated from healthy donor blood. M0: monocyte; N: neutrophil; MA1 and MA2 macrophage type 1 (pro- inflammatory and anti-tumoral) and 2 (pro-tumoral and anti-inflammatory). 12 μg of total protein (cell lysates) are loaded. The ponceau staining of these western blots is shown in Fig. S5. All samples are from the same donor. Experiment was repeated independently twice with similar results for blood from two healthy donors.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Comparison, Expressing, Isolation, Staining, Western Blot

Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 5. Poldip2 interaction with purified membrane fraction isolated from human neutrophils. Poldip2 (145 μl; 0.2 μM) and neutrophil membrane fractions (MF; 1.75 μl; 1.28 μM), containing the cytb558 (gp91phox/p22phox), were pre-incubated for 1 h with the cytb558/Poldip2 ratio of 1:20 (mol:mol). The MF and Polidp2 (alone) were treated similarly. The three samples were centrifuged at 190 000×g for 1h30. Pellet and supernatant were separated and analyzed by Western blot using antibodies raised against Nox2 (1:1500), p22phox (1:1500) and Poldip2 (1:1500). Experiments were performed at least three times with similar results.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Purification, Membrane, Isolation, Incubation, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Functional Peptide-Loaded Gelatin Nanoparticles as Eyedrops for Cornea Neovascularization Treatment

doi: 10.2147/IJN.S398769

Figure Lengend Snippet: Quantification of angiogenic cytokines from cornea protein lysate. Mice received chemical cauterization and were treated with PBS, gp91, and GNP-gp91 eye drops. Quantification of ( A ) Nox2, ( B ) VEGF, ( C ) MMP9, and ( D ) MMP2 levels from the cornea lysates. Data are presented as mean ± SD (n = 3), * p < 0.05, ** p < 0.001 compared with GNP-gp91 group.

Article Snippet: Mouse nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) ELISA Kit (My BioSource, cat no. MBS269961) was used to quantify Nox2 content in cornea.

Techniques: Eye Drops

Journal: Nature immunology

Article Title: NAD + salvage dependence due to ROS-mediated DNA damage in inflammatory macrophages

doi: 10.1038/s41590-019-0336-y

Figure Lengend Snippet: ( a ) Heatmap of genes expressed in response to ROS production and oxidative stress from of RNA-seq data of 1h polarized M0, M(γ+LPS), M(LPS) and M(IL-4). Statistically significant (adjusted p value < 0.1) up or down-regulated (> 2 fold) genes denoted by * in M(γ+LPS) polarized cells compared to M0 ( n = 3 biologically independent samples). ( b ) γ−H2AX expression in wild-type or Cybb −/− macrophages polarized for 1 h as indicated ( n = 3 biologically independent samples, representative of three independent experiments). Real time changes in OCR ( c ), basal OCR ( d ) and SRC ( e ) of macrophages unstimulated, or stimulated with γ+LPS for 0.5–4 h as indicated ( n = five technical replicates, representative of three independent experiments). ( f ) Flow cytometric analysis of MitoSOX staining in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h ( n = 3 biologically independent samples, representative of over ten independent experiments). Macrophages were treated with 10 mM n-acetylscysteine (NAC; g, h ) or MitoTempol (MT; i, j ) for 1 h, and subsequently left unpolarized (M0), or polarized with γ+LPS and MitoSox staining ( g, i ) and γ−H2AX expression ( h, j ) were assessed by flow cytometry ( n = 3 biologically independent samples, representative of four independent experiments). ( k ) 8-oxo-DG staining assessed by confocal microscopy in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 1 h. Scale bar: 5 μm, white arrows indicate nuclear co-localization of 8OHG staining, insert shows an enlargement of nuclei with 8OHG staining (data are representative of three independent experiments. Each experiment was performed with 3 biologically independent samples with 10 images collected per sample, per condition). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.

Article Snippet: C57BL/6 mice (RRID: IMSR_JAX:000664) and B6.129s-Cybb tm1Din /J mice (gp91phox) mice (RRID:ISMR_JAX:002365) were obtained from The Jackson Laboratory, and C57BL/6NRj (C57BL/6N) mice and C57BL/6JRj control mice were obtained from Janvier.

Techniques: RNA Sequencing, Expressing, Staining, Flow Cytometry, Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Construct, Transfection, Expressing, Western Blot, Control, Plasmid Preparation, Transduction, Knock-Out

Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A) EROS-FLAG affinity purification-mass spectrometry. Graph showing prey abundance (average number of peptide spectrum matches, n=4) by versus bait specificity (SP: SAINT probability score). The red line marks the SP score cut-off (0.9) for high confidence interacting proteins. The bait and interacting proteins relevant to this study are shown in red. (B) Network of protein interactions between EROS-interacting proteins derived from STRING. Coloured nodes represent proteins annotated with enriched GO terms relevant to this study. Red, “protein N-linked glycosylation”; blue, “T cell activation”; green, “positive regulation of glycoprotein biosynthetic process”, yellow “calcium ion transmembrane transport”. (C-D) EROS and gp91 phox localisation, following fixation and labelling with anti-EROS antibody, in HEK293 cells expressing STT3A-GFP and EROS constructs (C) or STT3A-GFP, gp91 phox -mRFP and EROS untagged constructs (D) . Graphs represents the intensity profile of STT3A-GFP and EROS signal or STT3A-GFP, gp91 phox -mRFP and EROS signal measured across the nuclear membrane (indicated in red line). (E) Expression of gp91 phox in HEK293 cells transfected with the indicated constructs and treated with OST inhibitor (NGI-1) at the indicated concentration. (F) Expression of gp91 phox in PLB985 cell line treated with NGI-1 at the indicated concentration. n= representative of 3 experiments.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Affinity Purification, Mass Spectrometry, Derivative Assay, Glycoproteomics, Activation Assay, Expressing, Construct, Membrane, Transfection, Concentration Assay

Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-C) P2X7 expression analysed by western blotting of macrophages isolated from control, EROS -/- (A) and gp91 phox -/- mice (B) and of control PLB985 cells and an EROS knock-out clone (C). (D-E) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector (D) and in HEK293 cells transiently expressing the specified constructs (E). (F-G) Interaction between EROS and P2X7 probed by immunoprecipitation of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot for P2X7 (F) and by NanoBIT assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector (G). (H) P2X1 expression in macrophages isolated from EROS -/- mice compared to control. n= 5 biological replicates. (I) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. n= representative of 3 independent experiments. See also Figure S3.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Expressing, Western Blot, Isolation, Control, Knock-Out, Plasmid Preparation, Construct, Immunoprecipitation, Cotransfection

Journal: Free radical biology & medicine

Article Title: Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration

doi: 10.1016/j.freeradbiomed.2023.03.001

Figure Lengend Snippet: A-C Rat midbrain neuron-glia cultures were challenged with vehicle or LPS (10 ng/ml; the concentration used in all in vitro experiments unless otherwise indicated). The expression of mRNA and protein of NOX2 (gp91 phox ) was measured by using RT-PCR (A) and Western blotting (B) with densitometry analysis (C) at indicated time points. Results are mean ± SEM of three experiments performed in triplicate (n = 3). * p < 0.05 compared with time-matched vehicle-treated controls. D, E Rat midbrain neuron-glia cultures were treated with vehicle or LPS for 6 days. D Representative immunohistochemical analysis showed that NOX2 was persistently upregulated during chronic neuroinflammation. E Representative images of double-labelled immunofluorescence showed increased expression of NOX2 (green) in neurons (red staining for neuronal maker Nissl) after LPS treatment. All immunoblotting and immunostaining images were representative of three independent experiments. Scale bars in (D) and (E) are 50 and 10 μm, respectively.

Article Snippet: The wildtype (WT) mice (C57BL/6J), B6.129S6-Cybbtm1Din/J (NOX2 −/− ; gp91 phox−/− ) mice, and timed-pregnant Fisher F344 rats were obtained from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Concentration Assay, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Immunofluorescence, Staining, Immunostaining

Journal: Free radical biology & medicine

Article Title: Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration

doi: 10.1016/j.freeradbiomed.2023.03.001

Figure Lengend Snippet: A-G. Rat neurons and microglia were separately cultured in a transwell system. This was achieved by seeding different amount of enriched microglia on the porous membrane of transwell inserts that were placed above neuronal layer grown in the well of the plate. At 24 hours after LPS or vehicle treatment of the co-cultures, neurons were collected for the measurement of mRNA or protein of subunits of NADPH oxidase. A Schematic diagram of the transwell cell culture system. B The mRNA expression of the catalytic subunit of NADPH oxidase, NOX1, NOX2 (gp91 phox ) and NOX4 isoforms in neurons was determined by using RT-PCR. C RT-PCR detected mRNA expression of cytosolic subunits (p47 phox and p67 phox ) in neurons. D The protein level of NOX2 and p67 phox in neurons was determined by Western blot. E The ratios of the densitometry values of NOX2 or p67 phox relative to GAPDH in ( C ) were normalized to vehicle-treated control. F Membrane and cytosolic fractions were isolated from neurons and used for examination of membrane translocation of cytosolic subunit p47 phox by Western blot. ATP1B3 and β-actin were used to monitor loading errors of membrane and cytosolic proteins respectively. G The densitometry values of p47 phox relative to ATP1B3 or β-actin in (F) were normalized to vehicle-treated control. H The mRNA expression of major subunits of NADPH oxidase (NOX2, p47 phox and p67 phox ) in N27 neuronal cell line was examined by RT-PCR after N27-microglia co-cultures grown in the transwell system were treated with LPS/vehicle for 24 hours. Data are shown as means ± SEM from three independent experiments performed in triplicate (B, C, E, G and H; n = 3). * p < 0.05 compared with the corresponding saline-treated controls.

Article Snippet: The wildtype (WT) mice (C57BL/6J), B6.129S6-Cybbtm1Din/J (NOX2 −/− ; gp91 phox−/− ) mice, and timed-pregnant Fisher F344 rats were obtained from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Activation Assay, Cell Culture, Membrane, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Isolation, Translocation Assay, Saline

Journal: Antioxidants

Article Title: Distinction between 2′- and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

doi: 10.3390/antiox10050723

Figure Lengend Snippet: Fluorescence enhancement of NS1-2 and NS1-3 upon binding to recombinant NOX2 C-terminal; the protein was complemented with FAD (at 1/1 ratio), Excitation at 460 nm, emission recorded in the range 480–700 nm. A broad maximum emission was detected at (560–580) nm. The data were corrected for the contribution of FAD by subtracting the signal obtained with FAD in buffer at each NOX2 concentration. The data are fitted to a dose–response curve embedded in Origin 8.1 software with the following parameters: NS1-2: A 1 (lower asymptote) = 0, A 2 (upper asymptote) = 212 ± 11, K D = 0.67 ± 0.11 µM, R 2 = 0.965; NS1-3: A 1 = 0, A 2 = 244 ± 14, K D = 1.14 ± 0.09 µM, R 2 = 0.984.

Article Snippet: The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech).

Techniques: Fluorescence, Binding Assay, Recombinant, Concentration Assay, Software

Journal: Antioxidants

Article Title: Distinction between 2′- and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

doi: 10.3390/antiox10050723

Figure Lengend Snippet: ( A ) NS1 fluorescence (excitation at 465 nm, emission at 620 nm) in selected organs of the mice after sacrifice at 4 h and 24 h post-IP relative to vehicle alone and their quantification. ( B ) NOX2 levels at 4 h in these organs determined by Western blots and their quantification relative to amino black presented in . The staining with amido black of the total proteins transferred onto the nitrocellulose membrane is used to normalize the levels of the protein of interest (i.e., NOX2) in each tissue analyzed. This normalization was used because any housekeeping proteins, such as β-actin or β-tubulin, show variable levels between tissues. Statistical significance indicated by stars (** p -value < 0.01) was evaluated by a two-way ANOVA followed by Tukey’s multiple comparisons test. A significant accumulation of NS1 was observed in the kidney after 4 h post-injection while at 24 h post-injection, NS1 fluorescence was significantly increased in the pancreas. No significant fluorescence of NS1 was noted in all other tissues analyzed.

Article Snippet: The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech).

Techniques: Fluorescence, Western Blot, Staining, Membrane, Injection

Journal: Antioxidants

Article Title: Distinction between 2′- and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

doi: 10.3390/antiox10050723

Figure Lengend Snippet: ( A ) Effect of 50 μM NS1 (mixture of isomers), NS1-2, NS1-3 on the migration of SUM159 breast cancer cells, see also ; ( B ) Western blot analysis of NOX2 in SUM159 cells treated or not with NS1 (50 µM) and their quantification after 48 h treatment; ( C , D ) Representative Western blots of NOX2, NOX1, NOX3 and NOX5 in SUM159 cells treated or not with NS1 or its isomers for 48 h and their quantification.

Article Snippet: The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech).

Techniques: Migration, Western Blot

Journal: Antioxidants

Article Title: Distinction between 2′- and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

doi: 10.3390/antiox10050723

Figure Lengend Snippet: Effect of NS1 and its isomers on ( A ) eNOS activity determined by the hemoglobin assay ; ( B ) Representative absorption changes around 340 nm due NOX2-induced NADPH consumption using NOX2 = 2.5 µM reconstituted with 50 µM FAD, NADPH = 50 µM with or without 25 µM of NS1-2 or NS1-3 in 20 mM Tris buffer pH = 7.5, 150 mM NaCl.

Article Snippet: The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech).

Techniques: Activity Assay

Journal: Antioxidants

Article Title: Distinction between 2′- and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

doi: 10.3390/antiox10050723

Figure Lengend Snippet: Comparison of the binding of NS1-2 ( A , shown in cyan) and NS1-3 ( B , shown in yellow) to a model of NOX2. For clarity, only FAD, and residues involved in the ligands binding are shown. ADE stands for adenine. ( C ) Zoom-in of the phosphate interaction with lysine/arginine residues, driving the recognition of NOS and NOX.

Article Snippet: The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech).

Techniques: Comparison, Binding Assay

Journal: The Journal of Physiological Sciences : JPS

Article Title: Role of purinergic activation and TRPC3 channels in the Frank-Starling mechanism

doi: 10.1016/j.jphyss.2025.100052

Figure Lengend Snippet: Involvement of extracellular ATP released via pannexin-1 in stretch-induced ROS production Effect of carbenoxolone (CBX), an inhibitor of pannexin-1, on stretch-induced changes in DCF slopes (n = 26, N = 3) ( A ). Effect of extracellular ATP administration in the presence of CBX (n = 31, N = 2) ( B ). Effect of extracellular ATP administration in TRPC3 −/− mice (n = 34, N = 2) ( C ), WT mice in the presence of Pyrazole 3 (Pyr3) (n = 29, N = 3) ( D ) and in NOX2 −/− mice (n = 36, N = 2) ( E ). Statistical significance was determined using repeated-measures one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: NOX2 knockout (NOX2 KO: NOX2 −/− ) mice (strain name: B6.129S- Cybb tm1Din /J) (stock # 002365) were purchased from the Jackson Laboratory, Bar Harbor, ME, USA.

Techniques:

Journal: The Journal of Physiological Sciences : JPS

Article Title: Role of purinergic activation and TRPC3 channels in the Frank-Starling mechanism

doi: 10.1016/j.jphyss.2025.100052

Figure Lengend Snippet: Schematic diagram of the mechanism of acute stretch-induced ROS production Acute myocardial stretch induces extracellular ATP release via pannexin-1, which activates P2Y receptors and induces PLC-mediated activation of the TRPC3-NOX2, resulting in increased ROS production. This stretch-induced ROS increases myocardial contractility and assists the Frank-Starling mechanism.

Article Snippet: NOX2 knockout (NOX2 KO: NOX2 −/− ) mice (strain name: B6.129S- Cybb tm1Din /J) (stock # 002365) were purchased from the Jackson Laboratory, Bar Harbor, ME, USA.

Techniques: Activation Assay