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: Translational stroke research

Article Title: MiR-126 Affects Brain-Heart Interaction after Cerebral Ischemic Stroke

doi: 10.1007/s12975-017-0520-z

Figure Lengend Snippet: Stroke significantly induces cardiac inflammation and oxidative stress compared to non-stroke mice. a ED1, b TGF-β, c NOX2 immunostaining and quantitative data, d Western blot assay

Article Snippet: For immunostaining, antibodies against mouse CD68 (ED1, a marker for monocytes/macrophages; 1:30, Bio-Rad); transforming growth factor (TGF-β; 1:500, Santa Cruz); monocyte chemotactic protein-1 (MCP-1; 1:100, Abcam); vascular cell adhesion molecule 1 (VCAM-1, 1:200, Santa Cruz); NADPH oxidase-2 (NOX2; 1:400, BD Bioscience) were employed.

Techniques: Immunostaining, Western Blot

Journal: Translational stroke research

Article Title: MiR-126 Affects Brain-Heart Interaction after Cerebral Ischemic Stroke

doi: 10.1007/s12975-017-0520-z

Figure Lengend Snippet: Stroke in miR-126EC−/− mice significantly increases inflammation and oxidative stress in the heart compared to WT miR-126fl/fl stroke mice. a NOX2 and b TGF-β immunostaining and quantitative data. c MiR-126 expression in serum and heart tissue. d Heart tissue VCAM-1 and MCP-1 gene expression measured by real-time PCR

Article Snippet: For immunostaining, antibodies against mouse CD68 (ED1, a marker for monocytes/macrophages; 1:30, Bio-Rad); transforming growth factor (TGF-β; 1:500, Santa Cruz); monocyte chemotactic protein-1 (MCP-1; 1:100, Abcam); vascular cell adhesion molecule 1 (VCAM-1, 1:200, Santa Cruz); NADPH oxidase-2 (NOX2; 1:400, BD Bioscience) were employed.

Techniques: Immunostaining, Expressing, Real-time Polymerase Chain Reaction

Journal: Translational stroke research

Article Title: MiR-126 Affects Brain-Heart Interaction after Cerebral Ischemic Stroke

doi: 10.1007/s12975-017-0520-z

Figure Lengend Snippet: MiR-126 regulates cardiomyocyte MCP-1 and VCAM-1 expression as well as regulates cardiomyocyte hypertrophy in primary cultured cardiomyocyte cell: a Sarcomeric a-actinin (a cardiomyocyte maker) shows that more than 95% cultured cells are cardiomyocyte. b MiR-126 expression in scrambled and miR-126 knockdown cardiomyocyte. c MCP-1, VCAM-1 and TGF-β, and NOX2 gene expression measured by real-time PCR. d Cardiomyocyte surface area measurements. Knockdown of miR-126 expression in cardiomyocyte increases cardiomyocyte size. e MiR-126EC−/− EC-Exo significantly decreases miR-126 expression compared to miR-126fl/fl-EC-Exo. f, g MiR-126fl/fl-EC-Exo treatment decreases, but miR-126EC−/−EC-Exo treatment fails to decrease cardiomyocyte hypertrophy compared to non-treatment control

Article Snippet: For immunostaining, antibodies against mouse CD68 (ED1, a marker for monocytes/macrophages; 1:30, Bio-Rad); transforming growth factor (TGF-β; 1:500, Santa Cruz); monocyte chemotactic protein-1 (MCP-1; 1:100, Abcam); vascular cell adhesion molecule 1 (VCAM-1, 1:200, Santa Cruz); NADPH oxidase-2 (NOX2; 1:400, BD Bioscience) were employed.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

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