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: International Journal of Molecular Sciences

Article Title: Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells

doi: 10.3390/ijms20071644

Figure Lengend Snippet: Bradykinin stimuli did not mediate NADPH oxidase 2, 4, or 5. Representative Phos-tag™ SDS-PAGE Western blots of glycosylated NOX2 and unglycosylated NOX2, NOX4, and NOX5. There was no phosphorylation of the core protein of each NOX by BK stimulation.

Article Snippet: Anti-NOX2, 3, and 5 antibodies were purchased from Proteintech.

Techniques: SDS Page, Western Blot