Journal: PLoS ONE

Article Title: Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

doi: 10.1371/journal.pone.0154157

Figure Lengend Snippet: ( A )–( B ), RT-PCR of NADPH-oxidases in 3T3 fibroblasts and MSC, respectively. Nox5 was not assessed in 3T3 fibroblasts, because it is absent in these cells . ( C )–( D ), 3T3 fibroblasts were stably infected by shRNAs to Nox4 or Duox1 and analyzed for corresponding mRNA ( C ) and protein expression ( D ). The graph shows mRNA expression levels normalized to those in cells expressing scrambled shRNA; (*) p < 0.05 as compared to scrambled controls in 3 independent experiments. ( E )–( F ), 3T3 fibroblasts were transiently transfected by siRNAs to Duox1 or Duox2 and analyzed for expression of mRNA in 3 independent experiments ( E ) and Nox4 and Duox1/2 proteins in 2 experiments ( F ). The mRNA expression levels were normalized to those in cells treated with non-targeting (NT) siRNA; (*) p < 0.05 as compared to NT controls. The western blots are typical of 2 experiments. ( G )–( H ), MSC were transiently transfected by siRNAs to Nox4, Duox1 or Duox2, and analyzed for mRNA in 3 independent experiments ( G ), and Nox4 protein expression in 2 experiments ( H ). The mRNA expression levels were normalized to those in NT controls; (*) p < 0.05 as compared to the NT controls. In this case Duox1/2 protein expression was not significantly altered by corresponding siRNAs (data not shown).

Article Snippet: Antibody to Nox4, Duox1 and Duox 2 were from Novus Biologicals (USA), antibody to vinculin were from Sigma (USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Infection, Expressing, shRNA, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

doi: 10.1371/journal.pone.0154157

Figure Lengend Snippet: Representative blots are shown on the left, statistics are shown on the right. The data are normalized to 10 min of stimulation in scrambled/NT controls for fibroblasts and 30 min of stimulation for MSC, which have therefore no error bars; (*) p < 0.05 as compared to scrambe ( scr ) or non-targeting ( NT ) controls from 2–4 independent experiments. ( A ) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in 3T3 fibroblasts stably expressing the indicated shRNAs. ( B ) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in 3T3 fibroblasts transiently transfected by indicated siRNAs. ( C ) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in MSC transiently transfected by indicated siRNAs. ( D ) Kinetics of cytoplasmic H 2 O 2 accumulation in 3T3 fibroblasts pre-treated by indicated siRNAs; PDGF added at 0 min. ( E ) Effects of Duox1/2 silencing on speed of 3T3 fibroblast migration. Shown are the results of shRNA- and siRNA-mediated silencing of Duox1/2 compared, respectively, to scramble and NT controls, which moved with identical speeds.

Article Snippet: Antibody to Nox4, Duox1 and Duox 2 were from Novus Biologicals (USA), antibody to vinculin were from Sigma (USA).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Transfection, Migration, shRNA

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Knockdown of Dual Oxidase 1 (DUOX1) Promotes Wound Healing by Regulating Reactive Oxygen Species (ROS) by Activation of Nuclear Factor kappa B (NF-κB) Signaling

doi: 10.12659/MSM.926492

Figure Lengend Snippet: The sequences of DUOX1 siRNA (siDUOX1).

Article Snippet: The membrane was then incubated with primary antibodies against DUOX1 (1: 1000, orb589688; Biorbyt), collagen I (1: 1000, Ab138492; Abcam), collagen III (1: 1000, Ab184993; Abcam), p21 (1: 2000, Ab109520; Abcam), p16 (1: 5000, Ab51243; Abcam), NF-κB (1: 2000, Ab16502; Abcam), H3 (1: 1000, Ab1791; Abcam), and GAPDH (1: 2000, #5174; Cell Signaling Technology [CST]) overnight at 4°C with gentle shaking.

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Knockdown of Dual Oxidase 1 (DUOX1) Promotes Wound Healing by Regulating Reactive Oxygen Species (ROS) by Activation of Nuclear Factor kappa B (NF-κB) Signaling

doi: 10.12659/MSM.926492

Figure Lengend Snippet: DUOX1 was highly expressed in slow-healing granulation tissue of burn wounds ( A ) Clinical burned wounds with fast healing (n=12) and slow healing (n=12) granulation tissue and normal tissue (n=6) were collected, and Q-PCR was used to detect the expression of DUOX1. Primary fibroblasts were isolated from fast-healing, slow-healing, and normal tissues. ( B ) Collagen I and III protein levels were detected by Western blotting. ( C ) Cell proliferation was detected by CCK8 at 0, 24, 48, and 72 h. * P <0.05, ** P <0.01, *** P <0.001 vs normal; # P <0.05, ## P <0.01 vs slow-healing. Normal tissues: skin tissue taken from the surface of traumatic injury, fast-healing granulation tissue: better recovery after 14 days’ treatment, slow-healing (n=12) granulation tissue: poor recovery after 14 days’ treatment.

Article Snippet: The membrane was then incubated with primary antibodies against DUOX1 (1: 1000, orb589688; Biorbyt), collagen I (1: 1000, Ab138492; Abcam), collagen III (1: 1000, Ab184993; Abcam), p21 (1: 2000, Ab109520; Abcam), p16 (1: 5000, Ab51243; Abcam), NF-κB (1: 2000, Ab16502; Abcam), H3 (1: 1000, Ab1791; Abcam), and GAPDH (1: 2000, #5174; Cell Signaling Technology [CST]) overnight at 4°C with gentle shaking.

Techniques: Expressing, Isolation, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Knockdown of Dual Oxidase 1 (DUOX1) Promotes Wound Healing by Regulating Reactive Oxygen Species (ROS) by Activation of Nuclear Factor kappa B (NF-κB) Signaling

doi: 10.12659/MSM.926492

Figure Lengend Snippet: Knockdown of DUOX1 significantly increased cell proliferation and inhibited the production of reactive oxygen species (ROS). ( A ) Following primary isolation from wound granulation tissue, fibroblasts (Vimentin-positive) were identified by immunohistochemical staining. ( B, C ) Primary fibroblasts were transfected with human DUOX1 interferon, and the interference efficiency of DUOX1 was detected by Q-PCR ( B ) and Western blot ( C ). Following DUOX1 gene interference in primary fibroblasts, ( D ) a CCK-8 kit was used to detect cell proliferation; ( E, F ) biochemical detection of cell supernatant MDA ( E ) and SOD ( F ) expression; ( G, H ) flow detection of ROS and ( G ) cell apoptosis ( H ); ( I ) Western blotting of NF-κB (nuclear and plasma), collagen, collagen III, P21, and P16. ** P <0.01, *** P <0.001 vs siNC. siNC – negative control of DUOX1 interference.

Article Snippet: The membrane was then incubated with primary antibodies against DUOX1 (1: 1000, orb589688; Biorbyt), collagen I (1: 1000, Ab138492; Abcam), collagen III (1: 1000, Ab184993; Abcam), p21 (1: 2000, Ab109520; Abcam), p16 (1: 5000, Ab51243; Abcam), NF-κB (1: 2000, Ab16502; Abcam), H3 (1: 1000, Ab1791; Abcam), and GAPDH (1: 2000, #5174; Cell Signaling Technology [CST]) overnight at 4°C with gentle shaking.

Techniques: Knockdown, Isolation, Immunohistochemical staining, Staining, Transfection, Western Blot, CCK-8 Assay, Expressing, Clinical Proteomics, Negative Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Knockdown of Dual Oxidase 1 (DUOX1) Promotes Wound Healing by Regulating Reactive Oxygen Species (ROS) by Activation of Nuclear Factor kappa B (NF-κB) Signaling

doi: 10.12659/MSM.926492

Figure Lengend Snippet: DUOX1-regulated wound healing, likely by regulation of reactive oxygen species (ROS). ( A, B ) Transfected primary fibroblasts with overexpression of human DUOX1 were constructed, and Q-PCR ( A ) and Western blot ( B ) were used to detect the efficiency of DUOX1 overexpression. Following pre-transfection of DUOX1 overexpression in primary fibroblasts for 24 h, cells were treated with NAC and ROS inhibitor (10 μM). ( C, D ) Expression of MDA ( C ) and SOD ( D ) were detected in cell supernatants; ( E, F ) flow cytometry was used to detect ROS and ( E ) cell apoptosis ( F ); ( G ) cell proliferation activity was detected using a CCK-8 kit; ( H ) expression of NF-κB (nuclear and plasma), collagen I, collagen III, P21, and P16 were detected by Western blotting. * P <0.05, ** P <0.01, *** P <0.001 vs vector or vehicle+vector; ## P <0.01, ### P <0.001 vs vehicle+oeDUOX1.

Article Snippet: The membrane was then incubated with primary antibodies against DUOX1 (1: 1000, orb589688; Biorbyt), collagen I (1: 1000, Ab138492; Abcam), collagen III (1: 1000, Ab184993; Abcam), p21 (1: 2000, Ab109520; Abcam), p16 (1: 5000, Ab51243; Abcam), NF-κB (1: 2000, Ab16502; Abcam), H3 (1: 1000, Ab1791; Abcam), and GAPDH (1: 2000, #5174; Cell Signaling Technology [CST]) overnight at 4°C with gentle shaking.

Techniques: Transfection, Over Expression, Construct, Western Blot, Expressing, Flow Cytometry, Activity Assay, CCK-8 Assay, Clinical Proteomics, Plasmid Preparation

Journal: Gastroenterology Research and Practice

Article Title: DUOX2 Expression Is Increased in Barrett Esophagus and Cancerous Tissues of Stomach and Colon

doi: 10.1155/2016/1835684

Figure Lengend Snippet: Primers sequence.

Article Snippet: After washing, the tissue sections were incubated in goat serum (Sigma, USA) for 20 min and then incubated overnight at 4°C with a rabbit polyclonal antibody specific for DUOX2 (Bioss, Beijing, China, Cat. number bs-11432R).

Techniques: Sequencing

Journal: Gastroenterology Research and Practice

Article Title: DUOX2 Expression Is Increased in Barrett Esophagus and Cancerous Tissues of Stomach and Colon

doi: 10.1155/2016/1835684

Figure Lengend Snippet: DUOX2 mRNA levels in Barrett esophagus, tumors of stomach, colon, and liver. (a) DUOX2 mRNA was at similar level in normal epithelium of esophagus and Barrett esophagus. The expressions of DUOX2 mRNA in gastric cancer and CRC were both significantly higher than that in each corresponding adjacent nonmalignant tissues. DUOX2 mRNA was hardly detected in hepatic carcinoma and adjacent nonmalignant liver tissue. ∗ P < 0.05. NE: normal esophagus; BE: Barrett esophagus; NG: adjacent nonmalignant gastric tissue; GC: gastric cancer; NC: adjacent nonmalignant colon tissue; CC: CRC; NH adjacent nonmalignant hepatic tissue; HC: hepatic carcinoma. (b) DUOX2 mRNA level in gastric carcinoma of patients with smoking history ( n = 8) was 7.9-fold higher than that in patients without smoking history ( n = 16). ∗ P < 0.001.

Article Snippet: After washing, the tissue sections were incubated in goat serum (Sigma, USA) for 20 min and then incubated overnight at 4°C with a rabbit polyclonal antibody specific for DUOX2 (Bioss, Beijing, China, Cat. number bs-11432R).

Techniques:

Journal: Gastroenterology Research and Practice

Article Title: DUOX2 Expression Is Increased in Barrett Esophagus and Cancerous Tissues of Stomach and Colon

doi: 10.1155/2016/1835684

Figure Lengend Snippet: DUOX2 detected by IHC in different tissues of esophagus, stomach, colon, and liver. In normal epithelium of esophagus DUOX2 protein was not detected by IHC (a1); in the columnar epithelium of Barrett esophagus the expression of DUOX2 was mainly located in the apical membrane of epithelial cell (a2); in adjacent nonmalignant gastric tissue epithelial cells were stained negatively with DUOX2 antibody; some unidentified cells were stained positively in lamina propria (b1, arrows); in gastric cancer DUOX2 was mainly found in cell plasma of tumor cells (b2); DUOX2 was at a low level and was mainly at the edge brush of epithelial cells, endocrine cells were strongly stained for DUOX2 (c1, arrows), and DUOX2 expression was higher in CRC; there were nuclei stained with DUOX2 antibody (c2). In hepatic carcinoma and its adjacent nonmalignant tissue there was no DUOX2 IHC staining (d1 and d2). Magnification: ×400.

Article Snippet: After washing, the tissue sections were incubated in goat serum (Sigma, USA) for 20 min and then incubated overnight at 4°C with a rabbit polyclonal antibody specific for DUOX2 (Bioss, Beijing, China, Cat. number bs-11432R).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Gastroenterology Research and Practice

Article Title: DUOX2 Expression Is Increased in Barrett Esophagus and Cancerous Tissues of Stomach and Colon

doi: 10.1155/2016/1835684

Figure Lengend Snippet: The relationship of DUOX2 expressions in gastric cancer and CRC with clinicopathological features.

Article Snippet: After washing, the tissue sections were incubated in goat serum (Sigma, USA) for 20 min and then incubated overnight at 4°C with a rabbit polyclonal antibody specific for DUOX2 (Bioss, Beijing, China, Cat. number bs-11432R).

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: NOX5 positively correlates with the progression of ESCC. a Immunohistochemical staining evaluated the expression of NOX1-5, or DUOX1, 2 in 92 pairs of ESCC, and their respective adjacent noncancerous tissues (cohort I). Representative results of immunohistochemical staining for NOX1-5, or DUOX1, 2 in the same set of consecutive tumor tissue and their respective adjacent noncancerous tissue slices. Magnification, ×10 as indicated. The chi-square test was employed to analyze correlations between tumors and their respective adjacent normal tissues. b Immunoblotting analysis of NOX5 in primary normal human esophageal epithelial cells (NEECs) and cultured ESCC cell lines or primary ESCC cells. GAPDH was used as a loading control. c Representative results of immunohistochemical staining for CA IX and NOX5 in the same set of consecutive tumor tissue slices (cohort I). High NOX5 levels significantly correlates with high level CA IX, examined using the chi-square test. d The chi-square test was employed to analyze correlations between different clinical parameters, including tumor stage, tumor status and lymph node status, without adjustments (ESCC: n = 95 biologically independent samples; cohort II). e Kaplan–Meier curves of ESCC patients with low versus high expression of NOX5 ( n = 95; HR = 4.181, 95% CI: 2.579–6.779, P < 0.0001, log-rank test). Significant differences were compared using the log-rank test (two-sided) without adjustments

Article Snippet: Antibody against NOX5 was from Abnova (Cat# PAB17793), antibodies against Ki-67 (Cat# 9027), CD31 (Cat# 3528), GAPDH(Cat# 5174), Flag (Cat# 2368), HA (Cat# 2367), c-Abl (Cat# 2862), Src (Cat# 2108) were from CST, antibodies against Pyk2 (Cat# ab226798), α1-ATPase (Cat# ab7671), phosphotyrosine (Cat# ab10321), DUOX2 (Cat# ab97266), Carbonic anhydrase IX (CA IX) (Cat# ab15086) were from Abcam, pSrc (Tyr 419 ) (Cat# 102-17936) was from Raybiotech, NOX1 (Cat# A8527), NOX2 (Cat# A1636), NOX3 (Cat# A3677), NOX4 (Cat# A11904), DUOX1 (Cat# A8583) were obtained from ABclonal, Src inhibitors-dasatinib (Cat# S1021) and PP2 (Cat# S7008) were purchased from Selleck Chemicals.

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Cell Culture

Journal: Medical Science Monitor

Article Title: Vitamin D Attenuates Hypoxia-Induced Injury in Rat Primary Neuron Cells through Downregulation of the Dual Oxidase 1 (DUOX1) Gene

doi: 10.12659/msm.925350

Figure Lengend Snippet: Figure 3. Downregulation of DUOX1 significantly attenuated hypoxia-induced injury in rat primary neuron cells. (A, B) The interference

Article Snippet: After blocking in 5% skim milk (BYL40422, BD Biosciences, Franklin Lakes, NJ, USA) for 1 hour at 25°C, the blots were probed with primary antibodies against HIF-1a (1: 400, Ab1, Abcam), DUOX1 (1: 1000, Orb539256, Biorbyt), VDR (1: 1000, Ab109234, Abcam), NF-kB (1: 2000, Ab16502, Abcam), cleaved caspase-3 (1: 1000, AF6311, Affinity), H3 (1: 1000, Ab1791, Abcam), and GAPDH (1: 2000, #5174, Cell Signaling Technology) overnight at 4°C with gentle shaking.

Techniques:

Journal: Medical Science Monitor

Article Title: Vitamin D Attenuates Hypoxia-Induced Injury in Rat Primary Neuron Cells through Downregulation of the Dual Oxidase 1 (DUOX1) Gene

doi: 10.12659/msm.925350

Figure Lengend Snippet: Figure 4. Vitamin D attenuated DUOX1-induced injury in rat primary neuron cells. (A, B) The overexpression efficiency of DUOX1 was

Article Snippet: After blocking in 5% skim milk (BYL40422, BD Biosciences, Franklin Lakes, NJ, USA) for 1 hour at 25°C, the blots were probed with primary antibodies against HIF-1a (1: 400, Ab1, Abcam), DUOX1 (1: 1000, Orb539256, Biorbyt), VDR (1: 1000, Ab109234, Abcam), NF-kB (1: 2000, Ab16502, Abcam), cleaved caspase-3 (1: 1000, AF6311, Affinity), H3 (1: 1000, Ab1791, Abcam), and GAPDH (1: 2000, #5174, Cell Signaling Technology) overnight at 4°C with gentle shaking.

Techniques: Over Expression

Journal: Frontiers in Pain Research

Article Title: Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia

doi: 10.3389/fpain.2024.1372942

Figure Lengend Snippet: All genes showing significant changes compared to the control condition (corrected for multiple t -tests).

Article Snippet: After blocking, the sections were double-labeled overnight at room temperature with primary antibodies: mouse anti DUOX1 (1:200, sc-393096, Santa Cruz Biotechnology, Inc), guinea pig anti NeuN (1;200, 266014, Synaptic Systems), a specific marker for neurons, or rabbit anti DUOX1 (1:200, PA585452, Invitrogen), mouse anti GFAP (1:200, G3893, Sigma), an antibody directed to glial fibrillary acid protein, a marker for satellite glia to determine localization of DUOX1 in different cell types.

Techniques:

Journal: Frontiers in Pain Research

Article Title: Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia

doi: 10.3389/fpain.2024.1372942

Figure Lengend Snippet: Genes that exhibited greater than ± 1.5 changes in fold regulations.

Article Snippet: After blocking, the sections were double-labeled overnight at room temperature with primary antibodies: mouse anti DUOX1 (1:200, sc-393096, Santa Cruz Biotechnology, Inc), guinea pig anti NeuN (1;200, 266014, Synaptic Systems), a specific marker for neurons, or rabbit anti DUOX1 (1:200, PA585452, Invitrogen), mouse anti GFAP (1:200, G3893, Sigma), an antibody directed to glial fibrillary acid protein, a marker for satellite glia to determine localization of DUOX1 in different cell types.

Techniques:

Journal: Frontiers in Pain Research

Article Title: Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia

doi: 10.3389/fpain.2024.1372942

Figure Lengend Snippet: Duox1 mRNA and DUOX1 protein expression TG. ( A ) RT-PCR analysis of Duox1 from TG of naive and CFA-inflamed rats. In CFA-treated rats, TG was analyzed one day after CFA administration in the masseter muscle. ** p < 0.05. Each group consisted of 4 animals, and data are shown as mean ± S.E.M ( B ) Immunoblots of DUOX1 and GAPDH in naïve and CFA-treated TG. ( C ) Averaged relative optical density (DUOX1/GAPDH) between naïve untreated and CFA-treated TG samples. ** p < 0.05. Each group consisted of three animals, and data are shown as mean ± S.E.M ( D ) The top row displays double labeling of DUOX1 with NeuN (a neuronal marker). DUOX1-positive elements were mainly detected in the cytoplasm of TG neurons. The bottom row shows double labeling with DUOX1 and GFAP (a marker for satellite glia). The neuropil stained positive for GFAP did not overlap with DUOX staining. Scale bar 25 μm.

Article Snippet: After blocking, the sections were double-labeled overnight at room temperature with primary antibodies: mouse anti DUOX1 (1:200, sc-393096, Santa Cruz Biotechnology, Inc), guinea pig anti NeuN (1;200, 266014, Synaptic Systems), a specific marker for neurons, or rabbit anti DUOX1 (1:200, PA585452, Invitrogen), mouse anti GFAP (1:200, G3893, Sigma), an antibody directed to glial fibrillary acid protein, a marker for satellite glia to determine localization of DUOX1 in different cell types.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Labeling, Marker, Staining

Journal: Frontiers in Pain Research

Article Title: Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia

doi: 10.3389/fpain.2024.1372942

Figure Lengend Snippet: Effects of FSS only. ( A,B ) Immunoblots of DUOX1 and β-actin and TRPA1 and β-actin from naïve and FSS treated rats. ( C,D ) Averaged relative optical density (DUOX1/β-actin and TRPA1/β-actin) from naïve and FSS treated rats. Each group consisted of five animals, and data are shown as mean ± S.E.M. ( E ) Changes in blood corticosterone levels in naïve and rats under FS stress were compared before and after the treatment. Blood samples from naïve rats were drawn at the same time for FS stress rats. Graphs represent percent changes between the two time points (mean ± SEM). * p < 0.05 (Mann-Whiney test).

Article Snippet: After blocking, the sections were double-labeled overnight at room temperature with primary antibodies: mouse anti DUOX1 (1:200, sc-393096, Santa Cruz Biotechnology, Inc), guinea pig anti NeuN (1;200, 266014, Synaptic Systems), a specific marker for neurons, or rabbit anti DUOX1 (1:200, PA585452, Invitrogen), mouse anti GFAP (1:200, G3893, Sigma), an antibody directed to glial fibrillary acid protein, a marker for satellite glia to determine localization of DUOX1 in different cell types.

Techniques: Western Blot