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: BMC Systems Biology

Article Title: Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase

doi: 10.1186/1752-0509-5-164

Figure Lengend Snippet: Correlation between protein and mRNA levels . Representative blots showing variation in (A) Prx2 and (B) Duox1 protein levels due to off-target effects of lentiviral shRNA. Correlation between protein levels derived from these blots and mRNA level for (C) Prx2 and (D) Duox1 in shRNA cell lines and wild type Jurkat cells.

Article Snippet: Primary antibody for Grx1 was purchased from R&D Systems (Minneapolis, MN), for full length Trx1 from BD Biosciences (Franklin Lakes, NJ), for Duox1 from Novus Biologicals (Littleton, CO), for Prx1 from Millipore, for Prx2 from Abcam (Cambridge, MA), for glutathione from Virogen (Watertown, MA), and for β-actin from Sigma-Aldrich.

Techniques: shRNA, Derivative Assay

Journal: BMC Systems Biology

Article Title: Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase

doi: 10.1186/1752-0509-5-164

Figure Lengend Snippet: PCR array target list

Article Snippet: Primary antibody for Grx1 was purchased from R&D Systems (Minneapolis, MN), for full length Trx1 from BD Biosciences (Franklin Lakes, NJ), for Duox1 from Novus Biologicals (Littleton, CO), for Prx1 from Millipore, for Prx2 from Abcam (Cambridge, MA), for glutathione from Virogen (Watertown, MA), and for β-actin from Sigma-Aldrich.

Techniques: Binding Assay

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

doi: 10.4049/jimmunol.1800443

Figure Lengend Snippet: FIGURE 5. DUOX1, DUOX2, and NOX2 are dispensable for BCR ligation–induced ROS production in the late phase and B cell proliferation. (A and B) RT-PCR assay (A) and Western blotting (B) for the expression of various NOX isoforms and their regulators in spleen B cells and BAL17 cells. The following tissues were used as control tissues in RT-PCR assay: colon for NOX1, NOXO1, and NOXA1; testis for NOX3; kidney for NOX4; and thyroid gland for DUOX1, DUOX2, DUOXA1, and DUOXA2. (C and D) Either CFSE-labeled (D) or unlabeled (C) spleen B cells from wild-type (WT), NOX22/2 and NOX22/2 DUOXA2/2 mice were stimu- lated with 10 mg/ml anti-IgM Ab for the indicated times (C) or 72 h (D). Fluorescence of DCFDA (C) or CFSE (D) were measured by flow cytometry. Fold changes in mean fluorescence intensity of DCFDA fluo- rescence compared with untreated cells were calculated (C). Alternatively, percentages of proliferated cells are indicated (D).

Article Snippet: Membranes were reacted by mouse anti-IkBa mAb (L35A5), rabbit anti–phospho-IkBa mAb (Ser32) (14D4), mouse anti-IkB kinase (IKK) b mAb (3G12), rabbit anti-IKKb mAb (D30C6), rabbit anti–phospho-IKKa/b mAb (Ser176/180) (16A6), rabbit anti–NF-kB p65 mAb (D14E12), rabbit anti–phospho-NF-kB p65 mAb (Ser536) (93H1), rabbit anti-CARD11 mAb (1D12), rabbit anti–phospho-CARD11 mAb, rabbit anti–phospho-Akt mAb (Ser473) (all from Cell Signaling Technology), anti–b tubulin (TUB 2.1) (Santa Cruz Biotechnology), rabbit anti-mouse p22phox Ab (Bioworld), rabbit anti-Nox1 Ab (GeneTex) or rabbit anti-Nox3 Ab (ABclonal Technology), mouse anti-mouse NOXO1 Ab (Santa Cruz Biotechnology), rabbit anti-mouse NOXA1 Ab (Bioss Antibodies), and rabbit anti-mouse DUOX2 Ab (Abcam), followed by a reaction with HRP-conjugated anti-rabbit IgG Ab (Invitrogen) or HRPconjugated anti-mouse IgG Ab (Cell Signaling Technology).

Techniques: Ligation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Labeling, Fluorescence, Cytometry