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

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