Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 1 Construction of the chimeric MGMT-Apn1 construct. The MGMT and Apn1 cDNA sequences were amplified from bacterial clones and the chimeric molecule was created using the overlapping PCR technique.3 The fusion product was subcloned into the pcDNA 3.0 vector, sequenced and used to transfect K562 cells.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Construct, Amplification, Clone Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 2 Analysis of K562 cells containing chimeric transgene. (a) The MGMT-Apn1 K562 clone and pcDNA vector-transfected control cells were analyzed by Northern blot analysis. Both the radiolabeled MGMT and Apn1 cDNAs hybridized with a 1.75 kb message from total RNA of MGMT-Apn1-transfected cells, but not of vector-control cells. The radiolabeled GAPDH cDNA hybridized to a 1 kb transcript from total RNA of both control cells and MGMT-Apn1-transfected cells. (b) Western blot analysis: The MGMT monoclonal antibody detected the 64 kDa MGMT- Apn1 fusion protein of transfected K562 cells, but not of control cells (lane 3). The antibody also detected the 21 kDa MGMT protein from MGMT- transfected K562 cells (lane 2). (c) MGMT activity analysis: The 18-mer O6-methyl guanine oligonucleotide assay was performed with 50 mg of protein from sonicated K562 cell extract. MGMT activity was detected in both MGMT- and MGMT-Apn1-transfected K562 cells. Presence of the 8-mer fragment indicates MGMT activity. (d) AP endonuclease activity assay: The 26-mer oligonucleotide assay was performed with 5 mg of total protein from sonicated cell extract in the presence of 20 mM EDTA. Apurinic endonuclease activity was detected in MGMT-Apn1-transfected cells, but not control or MGMT-transfected cells. Presence of the 14-mer indicates cleavage of the artificial AP site. Apn1 is Mg2+ independent, EDTA resistant whereas the endogenous mammalian Ape1 protein requires Mg2+ and is inactive in the presence of EDTA.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Plasmid Preparation, Transfection, Control, Northern Blot, Western Blot, Activity Assay, Oligonucleotide Assay, Sonication

Journal: Carcinogenesis

Article Title: Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells.

doi: 10.1093/carcin/bgn012

Figure Lengend Snippet: Fig. 5. Histone modifications at the promoter region of MGMT gene. The quantitative-ChIP assay was performed to analyze histone modifications including H3K9ac, H3K9me2 and H4ac at the promoter of MGMT gene. The data were expressed as mean ± SEM, representing the relative levels of specific histone modification state at region F3 of MGMTafter chromatin immune precipitation with specific antibodies against H3K9ac, H3K9me2 or H4ac with normalization by total input DNA. The fold differences compared with the control were calculated and shown in the figures. The states of different histone modifications in NiS- treated cells (NiS2) and NiS-transformed cells (NSTC2) (A) or NSTC2 cells treated with DAC or TSA (B) or NiS2 cells treated with DAC or TSA (C) were depicted. (D) Representative images resulted from Q-PCR assay at region F3, F4 of MGMT locus after ChIP using specific antibodies indicated in NSTC2 cells with or without treatment with DAC or TSA. IgG was served as a negative control.

Article Snippet: Antibodies used include MGMT, MBD1 and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA), DNMT1 and methyl-CpG-binding protein 2 (MeCP2) (Sigma–Aldrich Company, St Louis, MO) and MBD2 (Upstate Biotechnology, Lake Placid, NY).

Techniques: Control, Transformation Assay, Negative Control

Journal: Carcinogenesis

Article Title: Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells.

doi: 10.1093/carcin/bgn012

Figure Lengend Snippet: Fig. 6. The binding of MBDs and DNMT1 to the promoter region of MGMT gene. quantitative-ChIP assay was performed to detect the binding of MBDs or DNMT1 to the CpGs of MGMT gene using specific antibodies against MeCP2, MBD2, MBD1 or DNMT1. The data were expressed as mean ± SEM, representing the relative levels of MGMT amplification at region F3 after chromatin immune precipitation with specific antibodies against MeCP2, MBD2, MBD1 or DNMT1 with normalization by total input DNA. The fold differences compared with the control were calculated and shown in the figures. The amount of MGMT CpGs binding in the MeCP2, MBD2, MBD1 and DNMT1 immune complexes at region 3 in NiS-treated cells (NiS2) and NiS-transformed cells (NSCT2) (A) or in NSCT2 cells treated with DAC or silenced by shDNMT1 (B). (C) Representative images resulted from Q-PCR assay at region F3, F4 of MGMT locus after ChIP assay using specific antibodies indicated in NSTC2 cells treated with DAC or silenced by shDNMT1 introduction.

Article Snippet: Antibodies used include MGMT, MBD1 and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA), DNMT1 and methyl-CpG-binding protein 2 (MeCP2) (Sigma–Aldrich Company, St Louis, MO) and MBD2 (Upstate Biotechnology, Lake Placid, NY).

Techniques: Binding Assay, Control, Transformation Assay

Journal: Biopolymers and Cell

Article Title: Correlation of mutagenesis level with expression of reparative enzyme O6-methylguanine DNA methyltransferase during establishment of cell lines in vitro

doi: 10.7124/bc.00083d

Figure Lengend Snippet: Fig. 6. Cnange of the expression of reparative enzyme MGMT in cont- rol cells and in cells, treated with medium M2: 1 – control; 2 – cells cul- tivation with M2; 3 – next passage after incubation with M2; 4 – positi- ve control (cell line U937); 5 – control to variant 6; 6 – 10 days after treatment; 7 – control to variant 8; 8 – 15 days after treatment; 9 – cont- rol to variant 10; 10 – 26 days after treatment with M2 medium

Article Snippet: We used monoclonal antibodies against MGMT («Novus Biologicals Littleton», USA) and secondary species-specific antibody conjugated with horseradish peroxidase («Jackson ImmunoResearch», USA).

Techniques: Expressing, Control, Incubation, Variant Assay

Journal: bioRxiv

Article Title: Cannabidiol as a Prophylactic Agent Against Glioblastoma Growth: A Preclinical Investigation

doi: 10.1101/2025.10.29.685440

Figure Lengend Snippet: A) Immunofluorescence staining of MGMT and Ki-67 in GBM tumors from mice pretreated with inhaled CBD for 14 days, 3 days, or placebo. Representative images show that MGMT and Ki-67 levels were significantly reduced in the 14-day CBD pretreatment group compared to both the 3-day CBD and placebo groups (Scale bar: 40 μm). B) Quantification of staining intensity revealed significantly lower levels of MGMT and Ki-67 in the 14-day CBD pretreatment group compared to the 3-day CBD and placebo groups (p < 0.001). These results suggest that CBD pretreatment reduces key biomarkers associated with chemotherapy resistance and tumor proliferation, potentially enhancing therapeutic sensitivity. C) To further validate the effect of CBD on tumor progression, we assessed MGMT expression levels using Western blotting. The blot shows increased MGMT expression in the GBM tumors of mice pretreated with placebo (lane 2) compared to the normal brain tissue used as a negative control (lane 4). Long-term CBD pretreatment (14 days, lane 1) significantly reduced MGMT expression in the tumor compared to acute CBD pretreatment (3 days, lane 3), corroborating the reduction observed in the immunofluorescence analysis.

Article Snippet: Membranes were probed simultaneously with anti-mouse MGMT monoclonal antibody (O6-methylguanine-DNA methyltransferase, Novus Biologicals USA, Cat# NB100-168SS) and β-actin antibody (Thermo Fisher) for 24 hours.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Negative Control