Journal: International Journal of Molecular Sciences

Article Title: Dual Oxidase 2 (DUOX2) as a Proteomic Biomarker for Predicting Treatment Response to Chemoradiation Therapy for Locally Advanced Rectal Cancer: Using High-Throughput Proteomic Analysis and Machine Learning Algorithm

doi: 10.3390/ijms232112923

Figure Lengend Snippet: ( A ) Machine learning approaches to measure the predictive power of proteins for CCRT response. ( B ) Immunohistochemical (IHC) validation of dual oxidase 2 (DUOX2) as a predictive biomarker for CCRT response (statistical significance, * p -value < 0.05).

Article Snippet: The monoclonal mouse anti-DUOX2 antibody (NB110-61576, Novus Biologicals, Centennial, CO, USA) was diluted to 1:100.

Techniques: Immunohistochemical staining, Biomarker Discovery

Journal: Cancer Biology & Medicine

Article Title: A truncated protein product of the germline variant of the DUOX2 gene leads to adenomatous polyposis

doi: 10.20892/j.issn.2095-3941.2020.0305

Figure Lengend Snippet: Pedigrees and variants of 3 unrelated families carrying DUOX2 variants. Pedigrees of family 1(A), 2(B), and 3(C). The genotype and phenotype information are displayed, +/− represents a heterozygous nonsense (c.1588A>T; p.K530X) variant in the DUOX2 gene, and −/− represents the wild type. (D) The four DUOX2 variants in these 3 families were validated by Sanger sequencing. Their locations are depicted in a corresponding lollipop plot of DUOX2 (created with IBS) and the conserved domains of the hDuox2 protein are also shown (E).

Article Snippet: The slides were incubated simultaneously with rabbit anti-DUOX2 (400–500 amino acids; 1:500 dilution; NB110-61576; Novus Biological, Centennial, CO, USA) and mouse anti-DUOX2 (628–680 amino acids; 1:500 dilution, sc-398681; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Variant Assay, Sequencing

Journal: Cancer Biology & Medicine

Article Title: A truncated protein product of the germline variant of the DUOX2 gene leads to adenomatous polyposis

doi: 10.20892/j.issn.2095-3941.2020.0305

Figure Lengend Snippet: The truncated protein, hDuox2 K530, is overexpressed in adenomas. (A) Immunohistochemistry (IHC) staining of hDuox2 with an antibody targeting the N-terminal 1–100 amino acids of human Duox2 protein in the adenoma and normal tissues from a DUOX2 p.K530X carrier. The expression of non-phospho (active) β-Catenin was also detected in these 2 samples by IHC analysis. (B) The positions of the amino acid sequence recognized by 2 different anti-Duox2 antibodies are marked. (C) Immunofluorescence staining of wild-type and truncated proteins of hDuox2 in adenomas and normal tissues from a DUOX2 p.K530X carrier as well as the tumor from a DUOX2 wild-type patient. AA, amino acid.

Article Snippet: The slides were incubated simultaneously with rabbit anti-DUOX2 (400–500 amino acids; 1:500 dilution; NB110-61576; Novus Biological, Centennial, CO, USA) and mouse anti-DUOX2 (628–680 amino acids; 1:500 dilution, sc-398681; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Immunohistochemistry, Expressing, Sequencing, Immunofluorescence, Staining

Journal: Cancer Biology & Medicine

Article Title: A truncated protein product of the germline variant of the DUOX2 gene leads to adenomatous polyposis

doi: 10.20892/j.issn.2095-3941.2020.0305

Figure Lengend Snippet: Overexpression of truncated protein, hDuox2 K530, promotes cell proliferation through the unfolded protein response. (A) Construction of the RKO cell line with stable expression of hDuox2 K530. (B) Growth curve of RKO-hDuox2 K530 and the control group. **** P < 0.0001 (C) Confocal microscopy with anti-6 × His-tag (red) and anti-calreticulin (green) in the RKO-hDuox2 K530 cell line. (D) Immunohistochemistry staining (100×) of GRP78 in the adenoma and normal tissues from a DUOX2 p.K530X carrier. EV, empty vector.

Article Snippet: The slides were incubated simultaneously with rabbit anti-DUOX2 (400–500 amino acids; 1:500 dilution; NB110-61576; Novus Biological, Centennial, CO, USA) and mouse anti-DUOX2 (628–680 amino acids; 1:500 dilution, sc-398681; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Over Expression, Expressing, Confocal Microscopy, Immunohistochemistry, Staining, Plasmid Preparation

Journal: Cancer Biology & Medicine

Article Title: A truncated protein product of the germline variant of the DUOX2 gene leads to adenomatous polyposis

doi: 10.20892/j.issn.2095-3941.2020.0305

Figure Lengend Snippet: Schematic diagram of truncated protein, hDuox2 K530, causing adenomatous polyposis. The diagram shows that the truncated protein, hDuox2 K530, is translated due to a nonsense variant (c.1588A>T) occurring in the DUOX2 gene. Therefore, this misfolded protein is retained in the endoplasmic reticulum and activates an unfolded protein response, which could induce abnormal cell proliferation.

Article Snippet: The slides were incubated simultaneously with rabbit anti-DUOX2 (400–500 amino acids; 1:500 dilution; NB110-61576; Novus Biological, Centennial, CO, USA) and mouse anti-DUOX2 (628–680 amino acids; 1:500 dilution, sc-398681; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Variant Assay

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Primer sequences for RT-qPCR and siRNA of DUOX2.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Sequencing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: DUOX2 expression in five PC cells and normal pancreatic duct cell. (a) RT-qPCR was performed to analyze DUOX2 mRNA expression. (b) Western blot was used to detect DUOX2 protein expression. Data were presented as mean ± SD.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Upregulation of DUOX2 expression in PC tissues. (a) The mRNA expression level of DUOX2 was detected in tumor samples and adjacent normal tissues. (b) Representative photographs at 100x and 400x magnifications in tumor samples and paired adjacent normal tissues. Data were presented as mean ± SD. ∗∗ P < 0.01.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: DUOX2 expression in PDAC and its association with clinicopathological features in the GEPIA website (data from TCGA and GTEx databases). (a) DUOX2 mRNA expression in GEPIA in tumor tissues and normal tissues. (b) The expression level of DUOX2 in all stages presented in violin pictures. (c) K-M survival curve showing the association of DUOX2 with overall survival. (d) K-M survival curve showing the association of DUOX2 with disease free survival. Data were presented as mean ± SD. ∗ P < 0.05.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: DUOX2 expression association with OS and Cox analysis clinicopathological features based on our data. (a) K-M survival curve revealing the expression of DUOX2 associated with overall survival. (b) Risk factors and (c) independent risk factors for overall survival in univariate and multivariate Cox regression analysis. Data were presented as mean ± SD; ∗ P < 0.05.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Associate of DUOX2 mRNA expression with clinicopathological characteristics.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Knockdown of DUOX2 expression in PC cells (PANC-1 and BxPC-3) suppresses cell motility and proliferation. The expression of DUOX2 at (a) mRNA and (b) protein level with siRNA of DUOX2 interference. PANC-1 and BxPC-3 cell migration, (c) invasion, and (d) proliferation with siRNA of DUOX2 interference. Data were presented as mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Knockdown, Expressing, Migration

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Prediction of TFs and DUOX2. (a) Each top 5 TFs positively and negatively regulating DUOX2. (b) ONECUT2, (c) ZBTB7B, (d) NR1I2, (e) ELF3, (f) TCF7L2, (g) TCFL5, (h) RAC3, (i) CBX1, (j) USF2, and (k) RPA2 correlation with DUOX2 mRNA expression. Data were presented as mean ± SD. ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Expressing

Journal: BioMed Research International

Article Title: DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer

doi: 10.1155/2021/6530298

Figure Lengend Snippet: Prediction of miRNAs and DUOX2. (a) Potential regulatory miRNAs of DUOX2 present in 3 prediction and validation websites. (b) hsa-miR-5193 and hsa-miR-1343-3p binding putative sites of DUOX2 3′UTR. Correlation between (c) hsa-miR-5193, (d) hsa-miR-1343, and DUOX2 mRNA expression. Kaplan-Meier curves showing the expression of (e) hsa-miR-5193 and (f) hsa-miR-1343-3p related with overall survival in TCGA.

Article Snippet: Finally, the samples were incubated overnight at 4°C with DUOX2 polyantibody (biorbyt, UK, Orb41406), followed by incubation with secondary antibodies.

Techniques: Biomarker Discovery, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells

doi: 10.1371/journal.pone.0170327

Figure Lengend Snippet: ( A ) T7 endonuclease 1(T7E1) assay. HeLa cells were transiently transfected with either pX459/NOX4 gRNA #1 or pX459/NOX4 gRNA #2, and genomic PCR products were analyzed by T7E1 assay (left panel). HeLa cells transfected with pX459/NOX4 gRNA’s were selected with puromycin and single colonies were isolated. The genomic PCR product of each clone was analyzed by T7E1 assay (right panel). Experiments were repeated three times with similar observations, and representative images are shown. ( B ) DNA sequence analysis showed the presence of the NOX4 mutation in clones #1–2, #1–5 and #1–6. The black arrows indicate the heterogeneous genomic DNA sequences in each cell lines. ( C ) CRISPR-Cas9 introduced the insertion and deletion (indel) mutation in the target sites. ( D ) Western blot of NOX4 knockout cell lines. Equal amount of HeLa control and NOX4 knockout cell lysates were probed with anti-NOX4 antibody. Experiments were repeated three times with similar observations, and representative data is shown. ( E ) NOX4 knockout did not influence NOX1, NOX2, NOX3, NOX5, DUOX1 and DUOX2 levels. Protein levels of NOX1, NOX2, NOX3, NOX5, DUOX1 and DUOX2 in parental HeLa cells and three clones of NOX4 knockout cells. ( F ) NOX4 knockout showed lower H 2 O 2 production. The levels of H 2 O 2 were measured with the Amplex Red assay in triplicate. The graph shows the average and the standard deviation (SD). Control vs knockout cells. *: P <0.005, **: P <0.0001.

Article Snippet: The antibody for NOX5 from Boster Biological Technology (Pleasanton, CA), the antibody for DUOX1 from GeneTex (Irvine, CA), the antibody for DUOX2 from Novus Biologicals (Littleton, CO), and the antibody for p22phox from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Transfection, Isolation, Sequencing, Mutagenesis, Clone Assay, CRISPR, Western Blot, Knock-Out, Control, Amplex Red Assay, Standard Deviation