Journal: Oxidative Medicine and Cellular Longevity

Article Title: Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Effects of hypoxia on giant phagocytes (G ϕ ) area and markers.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Effects of hypoxia on gp91- phox and p22- phox expression in giant phagocytes (G ϕ ). Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. For double immunofluorescence staining fixed cytospins were stained with rabbit anti-gp91- phox and mouse anti-p22- phox primary Abs (1/100) or the corresponding isotype controls (rabbit IgG and mouse IgG2) followed by 1/400 CF 488A goat anti-rabbit IgG (green) and CF 647 goat anti-mouse IgG (red) staining. Nuclei were stained with DAPI (blue). Representative data out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Expressing, Isolation, Cell Culture, Double Immunofluorescence Staining, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Expression of NADPH oxidase subunits in giant phagocytes (G ϕ ) and the effects of NAC on their expression. Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. NAC (20 μ M) was added to PMN cultures 10 min prior to exposing to N, IH, or SH. Equal volumes of DMSO were added as a negative control. Cytospins were prepared and analyzed by confocal microscopy (see Materials and Methods). The developed G ϕ were stained by double immunofluorescence: (a) for CD66b (red) and gp91- phox (green) in untreated and NAC-treated G ϕ and (b) for gp91- phox (green) and p22- phox (red) in untreated and NAC-treated G ϕ . Nuclei were stained with DAPI. Representative photomicrographs out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Expressing, Isolation, Cell Culture, Negative Control, Confocal Microscopy, Staining, Immunofluorescence

Journal: Experimental and Therapeutic Medicine

Article Title: Interleukin 32 participates in cardiomyocyte-induced oxidative stress, inflammation and apoptosis during hypoxia/reoxygenation via the NOD2/NOX2/MAPK signaling pathway

doi: 10.3892/etm.2022.11504

Figure Lengend Snippet: IL-32 regulates the NOD2/NOX2/MAPK signaling pathway. (A) The expression levels of the proteins related to the NOD2/NOX2/MAPK signaling were determined using western blotting. (B) The efficacy of NOD2 overexpressing plasmids was verified reverse transcription-quantitative PCR and (C) western blot assays. (D) The effects of NOD2 overexpression on the expression levels of the NOD2/NOX2/MAPK signaling pathway-proteins were assessed using western blotting. *** P<0.001 vs. control or Ov-NC, ### P<0.001 vs. H/R and &&& P<0.001 vs. H/R + siRNA-IL-32 + Ov-NC. IL, interleukin; NOD, nucleotide-binding oligomerization domain; NOX, NADPH oxidase; NC, negative control; H/R, hypoxia and reoxygenation; siRNA, small interfering RNA.

Article Snippet: Following incubation of the blots with primary antibodies [IL-32 (cat. no. 11079-1-AP; 1:1,000; ProteinTech Group, Inc.), phosphorylated (p-)p65 (cat. no. GTX133899; 1:1,000; GeneTex, Inc.), cyclooxygenase-2 (COX-2; cat. no. 12375-1-AP; 1:2,000; ProteinTech Group, Inc.), p65 (cat. no. GTX102090; 1:2,000; GeneTex, Inc.), Bax (cat. no. 50599-2-lg; 1:5,000; ProteinTech Group, Inc.), cleaved caspase 3 (cat. no. GTX03281; 1:1,000; GeneTex, Inc.), caspase 3 (cat. no. GTX110543; 1:1,000; GeneTex, Inc.), Bcl2 (cat. no. 26593-1-AP; 1:2,000; ProteinTech Group, Inc.), NOD2 (cat. no. GTX30694; 1:1,000; GeneTex, Inc.), NADPH oxidase 2 (NOX2; cat. no. 19013-1-AP; 1:1,000; ProTeintech Group, Inc.), p-ERK (cat. no. 28733-1-AP; 1:5,000; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; 1:2,000; ProteinTech Group, Inc.), β-actin (cat. no. 20536-1-AP; 1:5,000; ProteinTech Group, Inc.)] at 4˚C overnight, the strips were incubated at room temperature with an HRP-conjugated secondary antibody (cat. no. SA00001-2; 1:5,000; ProteinTech Group, Inc.) for 2 h. An ECL kit (GK10008; GlpBio) was used for visualization. β-actin was used for normalization and ImageJ software (v1.8.0; National Institutes of Health) was used for densitometry.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Binding Assay, Negative Control, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Interleukin 32 participates in cardiomyocyte-induced oxidative stress, inflammation and apoptosis during hypoxia/reoxygenation via the NOD2/NOX2/MAPK signaling pathway

doi: 10.3892/etm.2022.11504

Figure Lengend Snippet: IL-32 induces oxidative stress via the NOD2/NOX2/MAPK signaling pathway. (A) The effects of NOD2 overexpression on cell viability were evaluated using the Cell Counting Kit-8 assay. (B) The effects of NOD2 overexpression on LDH levels were evaluated using the LDH assay kit. The effects of NOD2 overexpression on the levels of (C) MDA, (D) SOD and (E) ROS were assessed using specific assay kits. ** P<0.01 and *** P<0.001 vs. control; ## P<0.01 and ### P<0.001 vs. H/R; & P<0.05, && P<0.01 and &&& P<0.001 vs. H/R + siRNA-IL-32 + Ov-NC. IL, interleukin; NOD, nucleotide-binding oligomerization domain; NOX, NADPH oxidase; LDH, lactate dehydrogenase; MDA, malondialdehyde; SOD, superoxide dismutase; ROS, reactive oxygen species; H/R, hypoxia and reoxygenation; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Following incubation of the blots with primary antibodies [IL-32 (cat. no. 11079-1-AP; 1:1,000; ProteinTech Group, Inc.), phosphorylated (p-)p65 (cat. no. GTX133899; 1:1,000; GeneTex, Inc.), cyclooxygenase-2 (COX-2; cat. no. 12375-1-AP; 1:2,000; ProteinTech Group, Inc.), p65 (cat. no. GTX102090; 1:2,000; GeneTex, Inc.), Bax (cat. no. 50599-2-lg; 1:5,000; ProteinTech Group, Inc.), cleaved caspase 3 (cat. no. GTX03281; 1:1,000; GeneTex, Inc.), caspase 3 (cat. no. GTX110543; 1:1,000; GeneTex, Inc.), Bcl2 (cat. no. 26593-1-AP; 1:2,000; ProteinTech Group, Inc.), NOD2 (cat. no. GTX30694; 1:1,000; GeneTex, Inc.), NADPH oxidase 2 (NOX2; cat. no. 19013-1-AP; 1:1,000; ProTeintech Group, Inc.), p-ERK (cat. no. 28733-1-AP; 1:5,000; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; 1:2,000; ProteinTech Group, Inc.), β-actin (cat. no. 20536-1-AP; 1:5,000; ProteinTech Group, Inc.)] at 4˚C overnight, the strips were incubated at room temperature with an HRP-conjugated secondary antibody (cat. no. SA00001-2; 1:5,000; ProteinTech Group, Inc.) for 2 h. An ECL kit (GK10008; GlpBio) was used for visualization. β-actin was used for normalization and ImageJ software (v1.8.0; National Institutes of Health) was used for densitometry.

Techniques: Over Expression, Cell Counting, Lactate Dehydrogenase Assay, Binding Assay, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Interleukin 32 participates in cardiomyocyte-induced oxidative stress, inflammation and apoptosis during hypoxia/reoxygenation via the NOD2/NOX2/MAPK signaling pathway

doi: 10.3892/etm.2022.11504

Figure Lengend Snippet: IL-32 regulates inflammation and apoptosis via the NOD2/NOX2/MAPK signaling pathway. (A) The effects of NOD2 overexpression on the expression levels of TNF-α, IL-1β and IL-6 were assessed using reverse transcription-quantitative PCR analysis. (B) The effects of NOD2 overexpression on the expression levels of p-p65, p65 and COX-2 were assessed using western blot analysis. (C) The effects of NOD2 overexpression on the induction of apoptosis were determined with the TUNEL assay. (D) The effects of NOD2 overexpression on the expression levels of the apoptosis-related proteins were determined using western blot analysis. *** P<0.001 vs. control; ### P<0.001 vs. H/R; & P<0.05 and &&& P<0.001 vs. H/R + siRNA-IL-32 + Ov-NC. IL, interleukin; NOD, nucleotide-binding oligomerization domain; NOX, NADPH oxidase; COX-2, cyclooxygenase 2; H/R, hypoxia and reoxygenation; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Following incubation of the blots with primary antibodies [IL-32 (cat. no. 11079-1-AP; 1:1,000; ProteinTech Group, Inc.), phosphorylated (p-)p65 (cat. no. GTX133899; 1:1,000; GeneTex, Inc.), cyclooxygenase-2 (COX-2; cat. no. 12375-1-AP; 1:2,000; ProteinTech Group, Inc.), p65 (cat. no. GTX102090; 1:2,000; GeneTex, Inc.), Bax (cat. no. 50599-2-lg; 1:5,000; ProteinTech Group, Inc.), cleaved caspase 3 (cat. no. GTX03281; 1:1,000; GeneTex, Inc.), caspase 3 (cat. no. GTX110543; 1:1,000; GeneTex, Inc.), Bcl2 (cat. no. 26593-1-AP; 1:2,000; ProteinTech Group, Inc.), NOD2 (cat. no. GTX30694; 1:1,000; GeneTex, Inc.), NADPH oxidase 2 (NOX2; cat. no. 19013-1-AP; 1:1,000; ProTeintech Group, Inc.), p-ERK (cat. no. 28733-1-AP; 1:5,000; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; 1:2,000; ProteinTech Group, Inc.), β-actin (cat. no. 20536-1-AP; 1:5,000; ProteinTech Group, Inc.)] at 4˚C overnight, the strips were incubated at room temperature with an HRP-conjugated secondary antibody (cat. no. SA00001-2; 1:5,000; ProteinTech Group, Inc.) for 2 h. An ECL kit (GK10008; GlpBio) was used for visualization. β-actin was used for normalization and ImageJ software (v1.8.0; National Institutes of Health) was used for densitometry.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, TUNEL Assay, Binding Assay, Small Interfering RNA, Negative Control

Journal: American Journal of Translational Research

Article Title: Nox2 contributes to reactive oxygen species-induced redox imbalance in cancer-induced bone pain

doi:

Figure Lengend Snippet: Expression and cellular localization of Nox2 in the ipsilateral spinal cord. A-C. Nox2 (green) double fluorescence labeling with NeuN (red) for neurons, GFAP (red) for astrocytes and Iba1 (red) for microglia in the spinal cord dorsal horn at day 21 after tumor cell implantation. Amplified pictures showed the co-localization of Nox2 (green) and NeuN (red), GFAP (red) or Iba1 (red). B. The results showed that Nox2 was upregulated in CIBP + DMSO group compared with sham + DMSO group. C. Repeated injection of APO (200 mg/kg, i.p.) for 5 consecutive days notably decreased the expression of Nox2. The results showed that Nox2 was co-localized mostly with microglia (yellow) and neurons (yellow). (***P < 0.001 compared with the sham + DMSO group. ###P < 0.001 compared with the CIBP + DMSO group. n = 3 in each group). Scale bar = 200 μm.

Article Snippet: Then, the sections were then incubated for 24 h at 4°C with primary antibodies: mouse-anti 8-OHdG antibody (1:100, ab 62623, Abcam, Cambridge, MA, USA), rabbit anti-Nox2 antibody (1:50; 19013-1-AP, Proteintech, Wuhan, China), with mouse anti-NeuN antibody (NeuN marker; 1:100; MAB377; Millipore), mouse anti-GFAP antibody (astrocytes marker; 1:300; 3670; Cell Signaling Technology), or goat anti-Iba1 antibody (microglia marker; 1:100; ab5076; Abcam).

Techniques: Expressing, Fluorescence, Labeling, Amplification, Injection

Journal: American Journal of Translational Research

Article Title: Nox2 contributes to reactive oxygen species-induced redox imbalance in cancer-induced bone pain

doi:

Figure Lengend Snippet: Time-course of in Nox2 expression in the spinal cord after tumor cells implantation. Western blot assay detected the time course of Nox2 expression in control and CIBP rats (n = 6 per group). Fold change for the density of Nox2 was standardized to GAPDH. The fold change of Nox2 in the control (sham) group was set at 1 for quantification. (*P < 0.05 compared with sham-operated group, n = 6 per group).

Article Snippet: Then, the sections were then incubated for 24 h at 4°C with primary antibodies: mouse-anti 8-OHdG antibody (1:100, ab 62623, Abcam, Cambridge, MA, USA), rabbit anti-Nox2 antibody (1:50; 19013-1-AP, Proteintech, Wuhan, China), with mouse anti-NeuN antibody (NeuN marker; 1:100; MAB377; Millipore), mouse anti-GFAP antibody (astrocytes marker; 1:300; 3670; Cell Signaling Technology), or goat anti-Iba1 antibody (microglia marker; 1:100; ab5076; Abcam).

Techniques: Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: Nox2 contributes to reactive oxygen species-induced redox imbalance in cancer-induced bone pain

doi:

Figure Lengend Snippet: Expression of Nox2 in the spinal cord. APO (i.p., 200 mg/kg once a day) or a mixture of DMSO, tween 80 and saline (2 ml, i.p.) was injected on five consecutive days (from day 14 to day 18). Western blot analysis showed that inoculation of Walker 256 carcinoma cells significantly increased the expression of Nox2. Repeated injection of APO (200 mg/kg, i.p.) for 5 consecutive days significantly reversed the increasing of Nox2 after the tumor cells implantation (***P < 0.001 compared with the sham + DMSO group. ###P < 0.001 compared with the CIBP + DMSO group. n = 6 in each group).

Article Snippet: Then, the sections were then incubated for 24 h at 4°C with primary antibodies: mouse-anti 8-OHdG antibody (1:100, ab 62623, Abcam, Cambridge, MA, USA), rabbit anti-Nox2 antibody (1:50; 19013-1-AP, Proteintech, Wuhan, China), with mouse anti-NeuN antibody (NeuN marker; 1:100; MAB377; Millipore), mouse anti-GFAP antibody (astrocytes marker; 1:300; 3670; Cell Signaling Technology), or goat anti-Iba1 antibody (microglia marker; 1:100; ab5076; Abcam).

Techniques: Expressing, Injection, Western Blot

Journal: American Journal of Translational Research

Article Title: Nox2 contributes to reactive oxygen species-induced redox imbalance in cancer-induced bone pain

doi:

Figure Lengend Snippet: (A) Nox2 immunolabeled surface area (laminae I-IV; as depicted in A) were quantified using Image J software. (B) NeuN immunolabeled surface area (laminae I-IV; as depicted in A) were quantified using Image J software. (C) GFAP immunolabeled surface area (laminae I-IV; as depicted in A) were quantified using Image J software. (D) Iba1 immunolabeled surface area (laminae I-IV; as depicted in A) were quantified using Image J software. (**P < 0.01, ***P < 0.001 compared with the sham + DMSO group. ###P < 0.001 compared with the CIBP + DMSO group. n = 3 in each group).

Article Snippet: Then, the sections were then incubated for 24 h at 4°C with primary antibodies: mouse-anti 8-OHdG antibody (1:100, ab 62623, Abcam, Cambridge, MA, USA), rabbit anti-Nox2 antibody (1:50; 19013-1-AP, Proteintech, Wuhan, China), with mouse anti-NeuN antibody (NeuN marker; 1:100; MAB377; Millipore), mouse anti-GFAP antibody (astrocytes marker; 1:300; 3670; Cell Signaling Technology), or goat anti-Iba1 antibody (microglia marker; 1:100; ab5076; Abcam).

Techniques: Immunolabeling, Software