mouse anti-human mgmt monoclonal antibody mt3.1  (Kamiya)

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase expression is enhanced in early stage gastric cancer. A: Representative images of immunohistochemistry staining for O 6 -methylguanine-DNA methyltransferase (MGMT) in early stage gastric tumor and normal tissues ( n = 19); B: The mRNA level of MGMT in early stage gastric tumor and adjacent normal tissues ( n = 19). The mRNA expression was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); C: MGMT mRNA and protein expression in normal gastric epithelial cells and cancer cells by quantitative real-time polymerase chain reaction and immunoblot assays. GAPDH was used to normalize MGMT expression. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. MG-C: MNNG-induced malignant transformed cell; MU-C: MNU-induced malignant transformed cell; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

Techniques: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Transformation Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase is downregulated in N-nitroso compound-induced gastric epithelial cell malignant transformation. A and B: O 6 -methylguanine-DNA methyltransferase (MGMT) mRNA and protein expression in transformed gastric epithelial cells induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)/N-methyl-N-nitroso-urea (MNU) for 1, 4, and 8 wk; C: Cell anchorage-independent growth on soft agar for subcolones of MNNG/MNU-induced cells. C1-28: Different subcolones of MNNG/MNU-induced cells; MGMT(+): MGMT expression is upregulated in these subcolones; MGMT(-): MGMT expression is downregulated or no-changed in these subcolones; D: Apoptosis assay of MNNG/MNU-transformed subcolones after doxycycline treatment. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

Techniques: Transformation Assay, Expressing, Apoptosis Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: DNA hypomethylation contributes to O 6 -methylguanine-DNA methyltransferase upregulation in cell malignant transformation. A: Luciferase reporter assay in control and N-nitroso compound-transformed cells using PGL3-O 6 -methylguanine-DNA methyltransferase ( MGMT ) promoter; B and C: Methylation specific polymerase chain reaction and bisulfite genomic sequence analysis of the DNA methylation level of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced transformed cells compared with control cells; D: Correlation of MGMT expression and DNA methylation level of MGMT promoter based on the CCLE database; E: ChIP assay with anti-DNMT1 and anti-H3K9Me3 and H3K4Me2 antibodies for analyzing the DNMT1 binding to the MGMT promoter and the H3K9Me3 and H3K4Me2 levels in the MGMT promoter. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. M: Methylated; U: Unmethylated; IgG: Immunoglobulin G.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

Techniques: Transformation Assay, Luciferase, Reporter Assay, Control, Methylation, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Expressing, Binding Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: Inhibition of O 6 -methylguanine-DNA methyltransferase contributes to the N-nitroso compound-induced cell malignant phenotype. A and B: Cell anchorage-independent growth on soft agar and cell colony formation of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced cells after O 6 -BG treatment; C: Cell anchorage-independent growth on soft agar of cells with O 6 -methylguanine-DNA methyltransferase (MGMT) knock-down; D: Knock-down efficiency of MGMT detected by Western blot; E and F: Cell anchorage-independent growth on soft agar and cell colony formation of MGMT overexpressing cells; G: The mRNA expression of MGMT in gastric endoscopic biopsy samples. The analyses were repeated three times, and the results are expressed as the mean ± SD. a,c P < 0.05. c P < 0.05, precancerous lesion and early cancer vs advanced cancer. EV: Empty vector; MGMT: MGMT overexpression; MGMT: O 6 -methylguanine-DNA methyltransferase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and used for immunohistochemistry.

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Plasmid Preparation, Over Expression

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase expression is enhanced in early stage gastric cancer. A: Representative images of immunohistochemistry staining for O 6 -methylguanine-DNA methyltransferase (MGMT) in early stage gastric tumor and normal tissues ( n = 19); B: The mRNA level of MGMT in early stage gastric tumor and adjacent normal tissues ( n = 19). The mRNA expression was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); C: MGMT mRNA and protein expression in normal gastric epithelial cells and cancer cells by quantitative real-time polymerase chain reaction and immunoblot assays. GAPDH was used to normalize MGMT expression. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. MG-C: MNNG-induced malignant transformed cell; MU-C: MNU-induced malignant transformed cell; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Transformation Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: O 6 -methylguanine-DNA methyltransferase is downregulated in N-nitroso compound-induced gastric epithelial cell malignant transformation. A and B: O 6 -methylguanine-DNA methyltransferase (MGMT) mRNA and protein expression in transformed gastric epithelial cells induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)/N-methyl-N-nitroso-urea (MNU) for 1, 4, and 8 wk; C: Cell anchorage-independent growth on soft agar for subcolones of MNNG/MNU-induced cells. C1-28: Different subcolones of MNNG/MNU-induced cells; MGMT(+): MGMT expression is upregulated in these subcolones; MGMT(-): MGMT expression is downregulated or no-changed in these subcolones; D: Apoptosis assay of MNNG/MNU-transformed subcolones after doxycycline treatment. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Transformation Assay, Expressing, Apoptosis Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: DNA hypomethylation contributes to O 6 -methylguanine-DNA methyltransferase upregulation in cell malignant transformation. A: Luciferase reporter assay in control and N-nitroso compound-transformed cells using PGL3-O 6 -methylguanine-DNA methyltransferase ( MGMT ) promoter; B and C: Methylation specific polymerase chain reaction and bisulfite genomic sequence analysis of the DNA methylation level of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced transformed cells compared with control cells; D: Correlation of MGMT expression and DNA methylation level of MGMT promoter based on the CCLE database; E: ChIP assay with anti-DNMT1 and anti-H3K9Me3 and H3K4Me2 antibodies for analyzing the DNMT1 binding to the MGMT promoter and the H3K9Me3 and H3K4Me2 levels in the MGMT promoter. The analyses were repeated three times, and the results are expressed as the mean ± SD. a P < 0.05; b P < 0.01. M: Methylated; U: Unmethylated; IgG: Immunoglobulin G.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Transformation Assay, Luciferase, Reporter Assay, Control, Methylation, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Expressing, Binding Assay

Journal: World Journal of Gastrointestinal Oncology

Article Title: O 6 -methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

doi: 10.4251/wjgo.v14.i3.664

Figure Lengend Snippet: Inhibition of O 6 -methylguanine-DNA methyltransferase contributes to the N-nitroso compound-induced cell malignant phenotype. A and B: Cell anchorage-independent growth on soft agar and cell colony formation of N-methyl-N’-nitro-N-nitrosoguanidine/N-methyl-N-nitroso-urea-induced cells after O 6 -BG treatment; C: Cell anchorage-independent growth on soft agar of cells with O 6 -methylguanine-DNA methyltransferase (MGMT) knock-down; D: Knock-down efficiency of MGMT detected by Western blot; E and F: Cell anchorage-independent growth on soft agar and cell colony formation of MGMT overexpressing cells; G: The mRNA expression of MGMT in gastric endoscopic biopsy samples. The analyses were repeated three times, and the results are expressed as the mean ± SD. a,c P < 0.05. c P < 0.05, precancerous lesion and early cancer vs advanced cancer. EV: Empty vector; MGMT: MGMT overexpression; MGMT: O 6 -methylguanine-DNA methyltransferase.

Article Snippet: Mouse monoclonal anti-human MGMT antibody (dilution, 1:1000) and mouse monoclonal anti-human GADPH antibody (dilution, 1:2000) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Plasmid Preparation, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Olaparib Is a Mitochondrial Complex I Inhibitor That Kills Temozolomide-Resistant Human Glioblastoma Cells

doi: 10.3390/ijms222111938

Figure Lengend Snippet: High MGMT protein expression does not account for TMZ resistance in the U373/U373-R isogenic human glioblastoma model. ( a ) O6-methylguanine DNA methyltransferase (MGMT) and β-actin (loading control) protein expression as determined by Western blotting in U373 and U373-R glioblastoma cells in the absence of TMZ, with pictures on the left and quantification on the right ( n = 4). ( b ) Clonogenic assays of the cells that were pretreated for 72 h with increasing concentrations of MGMT inhibitor O6-benzylguanine (O6BG). Displayed are the plating efficiency of the vehicle-treated cells at 72 h (left; n = 6); and the surviving fraction (right; n = 6–12), where the data are normalized to the number of corresponding vehicle-treated cells. ( c ) Number of cells 72 h after treatment with the indicated concentrations of TMZ in combination with 50 µM O6BG (O6BG50; left; n = 4) or 100 µM O6BG (O6BG100; right; n = 4). ( d ) Clonogenic assays of the cells that were pretreated for 72 h with increasing concentrations of TMZ and O6BG. Displayed are the plating efficiency of the cells that were treated ± O6BG (left; n = 6); and the surviving fractions of the cells that received O6BG50 (middle; n = 6) or O6BG100 (right; n = 5–6), where the data are normalized to the number of corresponding vehicle-treated cells. All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, ns: p > 0.05 versus the corresponding control; ### p < 0.005 for whole curve comparison; by Student’s t -test ( a , b left), two-way ANOVA with Sidak’s post hoc test ( b right, c , d middle, d right), or one-way ANOVA with Dunnett’s post hoc test ( d left).

Article Snippet: The primary antibodies were mouse monoclonals against human MGMT (Santa Cruz Biotechnology, Heidelberg, Germany; catalogue #sc-56157) and against β-actin (Sigma-Aldrich; catalogue #A5441).

Techniques: Expressing, Western Blot