Journal: Nutrients

Article Title: Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis

doi: 10.3390/nu14183791

Figure Lengend Snippet: HXT + VitE improves TGF-β-pro-fibrogenic phenotype by attenuating the SMAD/NOX2 pathway. ( A ) ROS levels assessed by using CM-H 2 DCFDA in LX-2 cells NT, treated with HXT + VitE, or treated with TGF-β alone or with HXT + VitE for 24 h. Data are expressed as the mean ± SD of at least five independent experiments. *** p < 0.001 vs. NT; §§§ p < 0.001 vs. TGF-β. ( B ) Representative images of SMAD2/3 (green) cellular localization by IF in LX-2 cells NT, treated with HXT + VitE, or treated with TGF-β alone or with HXT + VitE for 3 h. Nuclei were stained by Hoechst (blue). Magnification 60×. ( C ) NOX2 gene expression by qRT-PCR in LX-2 cells NT, treated with HXT + VitE, or treated with TGF-β alone or with HXT + VitE for 24 h. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 vs. NT; § p < 0.05 vs. TGF-β. ( D ) Quantitative analysis and representative immunoblot of NOX2 protein expression by WB in LX-2 cells NT, treated with HXT + VitE, or treated with TGF-β alone or with HXT + VitE for 24 h. GAPDH protein levels were used as loading control. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 vs. NT; §§ p < 0.01 vs. TGF-β.

Article Snippet: Primary antibodies used were: rabbit anti-NOX2 antibody (1:100 dilution; 19013-1-AP) (Proteintech Group Inc., Rosemont, IL, USA) and rabbit anti-GAPDH antibody (1:5000 dilution; 5174) (Cell Signaling Technology Inc., Danvers, MA, USA).

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

Journal: Nutrients

Article Title: Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis

doi: 10.3390/nu14183791

Figure Lengend Snippet: HXT + VitE improves WD-induced liver fibrosis in mice. Representative images of H&E ( A ) and Mason’s trichrome ( B ) staining in liver sections of WD and WD + HXT + VitE mice (20× magnification). Hepatic gene expression by qRT-PCR of α-SMA ( C ), COL1A1 ( D ), COL3A1 ( E ), and NOX2 ( F ) in WD and WD + HXT + VitE mice. Data are expressed as the mean ± SD of at least three independent experiments. ** p < 0.01 and *** p < 0.001 vs. WD.

Article Snippet: Primary antibodies used were: rabbit anti-NOX2 antibody (1:100 dilution; 19013-1-AP) (Proteintech Group Inc., Rosemont, IL, USA) and rabbit anti-GAPDH antibody (1:5000 dilution; 5174) (Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Staining, Expressing, Quantitative RT-PCR

Journal: Nutrients

Article Title: Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis

doi: 10.3390/nu14183791

Figure Lengend Snippet: PIIINP and NOX2 levels correlate with fibrosis. Heatmap showing the correlation among the levels of PIIINP, NOX2, and fibrosis at T1 in patients included in our study.

Article Snippet: Primary antibodies used were: rabbit anti-NOX2 antibody (1:100 dilution; 19013-1-AP) (Proteintech Group Inc., Rosemont, IL, USA) and rabbit anti-GAPDH antibody (1:5000 dilution; 5174) (Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques:

Journal: Nutrients

Article Title: Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis

doi: 10.3390/nu14183791

Figure Lengend Snippet: HXT + VitE treatment reduces the circulating levels of PIIINP and NOX2 in patients with NAFLD-related fibrosis. PIIINP ( A ) and NOX2 ( B ) circulating levels at T0, T1, and T2 in patients belonging to HXT + VitE or PLA group. Values are mean ± SD. The p values for the time-by-group interaction from repeated measures one-way ANOVA are shown.

Article Snippet: Primary antibodies used were: rabbit anti-NOX2 antibody (1:100 dilution; 19013-1-AP) (Proteintech Group Inc., Rosemont, IL, USA) and rabbit anti-GAPDH antibody (1:5000 dilution; 5174) (Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel NOX Inhibitor Treatment Attenuates Parkinson’s Disease-Related Pathology in Mouse Models

doi: 10.3390/ijms23084262

Figure Lengend Snippet: The compound-6 effectively inhibits NOX-1, -2 and -4, based on IC 50 analysis.

Article Snippet: The primary antibodies pertaining to each protein analyzed were diluted at 1:1000 (p-Ser-129-α-synuclein, Cell signaling, cat# 23706S; NOX-1,2 rabbit antibody, Proteintech 17772–1-AP and 19013–1-AP) and incubated at 4 °C overnight.

Techniques:

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Changes of vessel density and Nox2 in placentae for low (LBW) and normal (NBW) birth weight piglets. a. The birth weight of piglets in LBW and NBW groups (n = 19). b. H&E staining to show vessel structures in placentae; the black arrows indicate the placental vessels. Scale bar, 200 μm. c. Histograms indicating placental vascular density in each group (n = 19). d. CD31 staining to show the distribution of vessel density in placentae. Scale bar, 50 μm. (e) Histograms indicating CD31 fluorescence intensity in each group (n = 14). f-h. The ROS, MDA, and GSH levels in placentae (n = 18). i. Protein levels of Nox2 and CD31 in placentae. j. Histograms indicating Nox2 and CD31 protein levels in each group (n = 12). k. Immunofluorescence images showing that Nox2 is highly expressed in blood vessels with low CD31 immunofluorescence density. Scale bar, 50 μm. l. Histograms indicating Nox2 fluorescence intensity in each group. m, n. Correlation between placental vascular density or CD31 fluorescence intensity and Nox2 fluorescence intensity. Data represent the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Staining, Fluorescence, Immunofluorescence

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Nox2 regulates PVECs tube formation and migration. a-d. Western blotting analysis of Nox2 expression in PVECs with siRNA against or infected with adenovirus Nox2 for 48 h (n = 3). e, i. Representative images of tube formation by PVECs. PVECs were seeded at a density of 4 × 10 4 cells/well on a plate precoated with Matrigel and cultured for 6 h after transfection with control siRNA or Nox2 siRNA, or with Ad-RFP or Ad-Nox2 for 48 h (n = 5). Scale bars, 200 μm. Images of Transwell (g, k) or wound healing assay (m, o) in each group (n = 5). Scale bars, 100 μm. All data represent the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Migration, Western Blot, Expressing, Infection, Cell Culture, Transfection, Wound Healing Assay

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: VEGF-A mediates the effects of Nox2 on PVECs tube formation. a. The mRNA expression of angiogenesis-related factors. Cells were transfected with indicated siRNA for 48 h, followed by culture for additional 24 h (n = 5). b. Western blotting analysis of the protein levels of Nox2 and VEGF-A in PVECs (n = 3). Cells were transfected with indicated siRNA for 48 h, followed by culture for additional 6, 12, and 24 h (n = 3). c. Immunofluorescence analysis of VEGF-A in PVECs. Cells were transfected with indicated siRNA for 48 h, followed by culture for additional 24 h. d. The concentration of VEGF-A in culture media (n = 5). e. Dual-luciferase reporter gene assay of the VEGF-A promoter activity (n = 5). f. Western blotting analysis of Nox2 and VEGF-A expression levels in PVECs. Cells were co-transfected with si Nox2 and si VEGF-A for 48 h (n = 6). g. The representative images of tube formation of PVECs. Cells were co-transfected with si Nox2 and si VEGF-A for 48 h and seeded at a density of 4 × 10 4 cells/well on a plate precoated with Matrigel and cultured for 6 h (n = 5); scale bars, 200 μm. h, i. Western blotting analysis of Nox2 and p-VEGFR2 expression levels in PVECs with siRNA against Nox2 treated with or without bevacizumab (20 μg/mL) for 24 h. j. The representative images of tube formation of PVECs cultured with conditioned medium from the siRNA transfected cells treated with or without bevacizumab (20 μg/mL) for 6 h (n = 5); scale bars, 200 μm. k. The protein level of VEGF-A in PVECs under the treatment of Nox2 ds-tat (10 μM) (n = 3). l. The representative images of tube formation of PVECs with siRNA against VEGF-A treated with or without Nox2 ds-tat (10 μM) (n = 5); scale bars, 200 μm.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Expressing, Transfection, Western Blot, Immunofluorescence, Concentration Assay, Luciferase, Reporter Gene Assay, Activity Assay, Cell Culture

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Nox2 regulates STAT3 signaling pathway. (a, b) The protein levels of VEGE-A, p-STAT3 and STAT3 in placentae (n = 12). c-f. Western blotting analysis of p-STAT3 and VEGF-A expression in control and Nox2-knockdown or Nox2-overexpression cells (n = 3). g-i. Western blotting analysis of nuclear and cytoplasmic distribution of p-STAT3 in PVECs with Nox2 knockdown or overexpression; histone H3 and β-Actin serve as nuclear and cytoplasmic markers, respectively (n = 3). k, l. The mRNA expression levels of STAT3-targeted genes (n = 5). m-p. Protein overturn assay. PVECs with Nox2 silence or overexpression were incubated with 12 μg/mL CHX for the indicated interval; Western blotting was employed to observe the p-STAT3 protein levels (n = 3). q. Immunoprecipitation (IP) analysis of the interaction of Nox2 and STAT3. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Western Blot, Expressing, Over Expression, Incubation, Immunoprecipitation

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Nox2 regulates angiogenesis via activating the STAT3/VEGF-A signaling pathway. a. Western blotting analysis of Nox2, VEGF-A, and p-STAT3 protein levels in control and Nox2-knockdown cells treated with or without 5 μM Stattic (n = 3). b. Immunofluorescence analysis of VEGF-A and p-STAT3 in control and Nox2-knockdown cells treated with or without 5 μM Stattic. c. VEGF-A promoter activity (n = 5). d, e. Western blotting analysis of the VEGF-A protein level in PVECs (n = 3); cells were treated with 5 μM Stattic for 2 h, followed by stimulation with 10 μM Nox2 ds-tat for 24 h. f. The representative images of tube formation of PVECs transfected with si Ctrl or si Nox2 and treated with 5 μM Stattic for 6 h (n = 5); bar = 100 μm. g. The representative images of tube formation of PVECs treated with 10 μM Nox2 ds-tat and 5 μM Stattic (n = 5); bar = 100 μm.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Western Blot, Immunofluorescence, Activity Assay, Transfection

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Nox2 overexpression inhibits the activation of the STAT3 signaling pathway by inducing mitochondrial ROS production. a. Representative fluorescence images of MitoSOX and JC-1-stained cells; scale bar: 100 μm. b-d. GSH, mt DNA, and ATP contents (n = 6). e. The protein levels of PGC-1α and NRF1 (n = 3). f. The Nox2, VEGF-A, and p-STAT3 protein levels of PVECs transfected with Ad-Ctrl or Ad- Nox2 under the treatment of MitoQ (500 nM) for 24 h (n = 3). g. Immunofluorescence analysis of p-STAT3 in PVECs transfected with Ad-Ctrl or Ad-Nox2 under the treatment of MitoQ for 24 h. h. Representative images of tube formation and transwell assay of PVECs (n = 5).

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Over Expression, Activation Assay, Fluorescence, Staining, Transfection, Immunofluorescence, Transwell Assay

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Knockdown of Nox2 induces angiogenesis in Matrigel plugs in vivo . a. BALB/c nude mice were injected subcutaneously with 400 μL Matrigel mixed with 100 μL PVEC conditioned media (CM) for 15 days, then the plugs were excised. b. Body weight of BALB/c nude mice at day 1 and 15 in the trial. c. The Matrigel plugs were photographed (c), quantified for hemoglobin content (d), stained with CD31(e, upper) and hematoxylin and eosin (e, below). The CD31 immunofluorescence intensity (f) and cells (g) in the Matrigel plugs. Results are expressed as the mean ± SEM (n = 6).

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: In Vivo, Injection, Staining, Immunofluorescence

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet: Schematic diagram showing the role of Nox2-mediated VEGF-A dependent angiogenesis via STAT3 signaling. Nox2 deficiency decreased mitochondrial ROS generation, which in turn induced STAT3 activation, thereby increasing VEGF-A expression and excretion and promoting VEGF-A dependent autocrine angiogenesis.

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques: Activation Assay, Expressing

Journal: Redox Biology

Article Title: Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

doi: 10.1016/j.redox.2021.102051

Figure Lengend Snippet:

Article Snippet: After staining for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min, followed by incubation at room temperature overnight in a wet box with the following primary antibodies: Goat anti‐rabbit p-STAT3 (Abcam, USA), goat anti‐mouse VEGF-A antibody (Novus, USA), goat anti‐rabbit Nox2 (Proteintech, USA).

Techniques:

Journal: BMC Cardiovascular Disorders

Article Title: Role of peroxisome proliferators-activated receptor-gamma in advanced glycation end product-mediated functional loss of voltage-gated potassium channel in rat coronary arteries

doi: 10.1186/s12872-020-01613-y

Figure Lengend Snippet: Protein expressions in incubated RSCAs detected by Western blot. The RSCAs were incubated with DMEM, 200 μg/mL BSA, 200 μg/mL AGE, 200 μg/mL AGE+ 1 mM ALA, 200 μg/mL AGE+ 0.1 mM PIO, or 200 μg/mL AGE + 0.1 mM PIO + 0.1 mM GW9662 for 24 h. a Representative blots for RAGE, Kv1.2, Kv1.5, PPAR-γ and NOX-2. β-actin was used as an internal reference. b-d Mean data for protein expression levels. n = 3 per group. * P < 0.05 vs. BSA; # P < 0.05 vs. AGE; § P < 0.05 vs. AGE + PIO

Article Snippet: Equal amounts of protein were separated by 10% SDS-PAGE, transferred to PVDF membranes, and stained with the following primary antibodies (all from Abcam, Cambridge, UK): polyclonal rabbit anti-RAGE (1:500), monoclonal mouse anti-Kv1.2 (1:1000), monoclonal mouse anti-Kv1.5 (1:1000), monoclonal rabbit anti-PPAR-γ (1:1000), monoclonal rabbit anti-NOX-2 (1:1000), and anti-β-actin (1:10000), followed by incubation with HRP-conjugated goat anti-mouse IgG antibody (1:20000, Proteintech Group Inc., IL, USA).

Techniques: Incubation, Western Blot, Expressing

Journal: BMC Cardiovascular Disorders

Article Title: Role of peroxisome proliferators-activated receptor-gamma in advanced glycation end product-mediated functional loss of voltage-gated potassium channel in rat coronary arteries

doi: 10.1186/s12872-020-01613-y

Figure Lengend Snippet: Protein expressions in cultured CSMCs detected by Western blot. The CSMCs were incubated with DMEM, 100 μg/mL BSA, 100 μg/mL AGE, 100 μg/mL AGE+ 10 μM ALA, 100 μg/mL AGE+ 1 μM PIO, or 100 μg/mL AGE+ 1 μM PIO + 1 μM GW9662 for 24 h. a Representative blots for RAGE, Kv1.2, Kv1.5, PPAR-γ and NOX-2. β-actin was used as an internal reference. b-d Mean data for protein expression levels. n = 3 per group. * P < 0.05 vs. BSA; # P < 0.05 vs. AGE; § P < 0.05 vs. AGE + PIO

Article Snippet: Equal amounts of protein were separated by 10% SDS-PAGE, transferred to PVDF membranes, and stained with the following primary antibodies (all from Abcam, Cambridge, UK): polyclonal rabbit anti-RAGE (1:500), monoclonal mouse anti-Kv1.2 (1:1000), monoclonal mouse anti-Kv1.5 (1:1000), monoclonal rabbit anti-PPAR-γ (1:1000), monoclonal rabbit anti-NOX-2 (1:1000), and anti-β-actin (1:10000), followed by incubation with HRP-conjugated goat anti-mouse IgG antibody (1:20000, Proteintech Group Inc., IL, USA).

Techniques: Cell Culture, Western Blot, Incubation, Expressing