Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 1. TMZ sensitivity and MGMT and miR-221/222 expression in glioma cells. (A) Glioma cells were treated with TMZ (300µMol) for 24 hr. Cell viability was evaluated with an MTT assay. (B) Western blot analysis of MGMT expression in glioblastoma cells. (C) Real time PCR of miR-221 expression in glioblastoma cells. (D) RNA Hybrid prediction analyzes of miR-222, miR-221, and MGMT 3’ UTR. In bold are shown the mutated oligonucleotides. Luciferase activity of HEK-293 cells transiently co-transfected with the luciferase reporter containing wild-type MGMT-3’UTR or mutant MGMT-3’UTR in the presence of pre-miR-222, miR-221, or scrambled oligonucleotide. Representative of at least three independent experiments. *** p<0.001 versus control, ** p<0,0037 versus control.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Expressing, MTT Assay, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Mutagenesis, Control

Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 2. miR-221/222 target MGMT. (A) Western blot analysis and real time PCR of MGMT protein and RNA after miR-221/222 transfection of T98G cells. (B) Western blot analysis and real time PCR of MGMT protein and RNA after anti-miR-221 and -222 transfection of U87MG cells. (C) Western blot of MGMT expression upon miR-221 transfection of LN428 cells. (D) Western blot analysis of MGMT expression in T98G cells, as a control, and the melanoma cell line A375 upon miR-221 transfection. (E) Analysis of methylation status of MGMT promoter in T98G and U87MG upon miR- or anti- miR-221/222 transfection. U is for the un-methylated form, M for methylated form, NL is for normal lymphocytes, used as control.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Expressing, Control, Methylation

Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 3. miR-221 modulates TMZ sensitivity. (A) Cell viability of T98G, LN428, and A375 cells transfected with miR-221 and miR-222 upon TMZ treatment (300 µMol) for 24 hrs. **p value<0.0082 versus scr column, ***p value<0.005 versus scr column. (B) Growth curve of T98G and LN428 cells transfected or not with miR-221 after 24 hrs of treatment with TMZ. (C) Colony assay of T98G and LN428 cells transfected with miR-221 and then treated for 24 hrs with TMZ (300 µMol). Cells were left to grow for 6 days after treatment removal. (D) MGMT expression rescues cell viability after TMZ treatment in T98G and LN428 cells overexpressing miR-221 **p value<0.0082 versus untransfected MGMT column. (E) Correlation between miR-221 expression and TMZ sensitivity in nine primary glioblastoma cell lines and in six glioblastoma cell lines.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Transfection, Colony Assay, Expressing

Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 4. miR-221 promotes DNA damages upon TMZ treatment. (A) Apoptotic cell death assessed by FACS in T98G cells transfected with miR-221 or scrambled sequence and MGMT and treated with TMZ for 24 hrs. *** p value< 0.005 versus untrasfected MGMT column. (B) Active caspase-3 quantification in T98G cells as indicated and treated with TMZ for 24 hrs in the presence or absence of 3 hrs pre-treatment with ZVAD-fmk. (C) Upper panel Time course analysis of caspase-3 activation upon TMZ treatment in T98G cells transfected with miR-221 or with scrambled sequence. Lower panel Western blot analysis of caspase-3 activation after miR-221 and MGMT transfection. (D) Cell viability of T98G cells transfected with miR-221 or with scrambled sequence treated with TMZ for 24 hrs in the presence or absence of 3 hrs pre-treatment with ZVAD-fmk. ** p value< 0.0034 versus only treated TMZ column, Student’s t test.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Transfection, Sequencing, Activation Assay, Western Blot

Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 5. miR-221 promotes DNA damage. (A) Alkaline comet assay of T98G cells transfected with miR-221 and treated with TMZ for the indicated times. (B) Analysis of γH2AX in T98G cells transfected with scrambled control miR or miR-221, treated with TMZ in the presence or in the absence of MGMT cDNA, by immunocytofluorescence (upper and medium panel) or by Western blot (lower panel). (C) Western blot analysis of the indicated proteins upon transfection of T98G cells with miR-221 and MGMT cDNA and TMZ treatment for 24 hrs.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Alkaline Single Cell Gel Electrophoresis, Transfection, Control, Western Blot

Journal: PloS one

Article Title: MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Figure 6. Association of miR-221 and MGMT expression. Mann–Whitney U test analysis was performed to evaluate the association between miR-221 and MGMT expression in long- and short -survival groups of patients. The expression of miR-221 (2^-Dct) (A-B) and MGMT (2^-Dct) are inversely correlated with patient survival (p < 0.0490 and p = 0.043, respectively).

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Expressing, MANN-WHITNEY

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: Differential expression of BC200 RNA in GB cell lines. ( A ) The differential expression of BC200 RNA in GB cell lines and normal human astrocytes are shown. ( B ) The viability of GB cells was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( C ) Flow cytometry analysis of the ALDH1 + /CD133 + portion in GB cell lines and normal human astrocytes. ( D ) The level of BC200, BCRP1, MDR1, MRP1 and MGMT in GB cell lines was analyzed using RT-qPCR. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: Quantitative Proteomics, Sulforhodamine B Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: BC200 RNA promotes TMZ resistance in GB through sponge miR-218-5p. ( A ) The viability of shBC200 and OEBC200 GB cell lines was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( B ) The levels of MGMT, BCRP1, MDR1, and MRP1 following shBC200 and OEBC200 in GB cells were determined through western blot. ( C ) MicroRNA profiling analyses showed that shBC200 and OEBC200 contained high and low levels of miR-218-5p, respectively. ( D ) LncBase Predicted v.2 predicted that a high binding score of miR-218-5p with BC200. ( E ) BC200 directly interacts with multiple binding sites to hsa-miR-218-5p. *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: Sulforhodamine B Assay, Western Blot, Binding Assay

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: miR-218-5p regulated cell sphere formation, colony formation, and TMZ resistance in GB cells in vitro. ( A ) The differential expression of miR-218-5p in GB cell lines and normal human astrocytes. ( B ) Pearson’s correlation curve identified a negative correlation between BC200 and miR-218-5p in GB tissues. ( C ) Sphere formation assays showed that inhibition or mock transfection of miR-218-5p regulated GB cell stemness. ( D ) Colony formation assays showed that inhibition or mock transfection of miR-218-5p regulated GB cell survival. ( E ) The viability of GB cells with inhibition or mock transfection of miR-218-5p was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( F ) CCK-8 assay showed that inhibition or mock transfection of miR-218-5p had no effect on GB cell proliferation. ( G ) The protein levels of SOX2, Oct4, BRPC1, MRP1 and MDR1 with inhibition or mock transfection of miR-218-5p. ( H ) The protein levels of MGMT, MLH1, MSH2, MSH6 and PMS2 following inhibition or mock transfection of miR-218-5p in GB cells were determined through western blot. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: In Vitro, Quantitative Proteomics, Inhibition, Transfection, Sulforhodamine B Assay, CCK-8 Assay, Western Blot

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: The lncRNA BC200 RNA sponges miR-218-5p regulated MGMT and MMR system enhancing self-renewal and TMZ resistance of GBM cells.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques:

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 2. ERp29 modulates the expression of tumour suppressors and pro-oncogenes by epigenetic regulation. (a) ERp29 expression promoted/inhibited promoter demethylation of tumour suppressors/pro- oncogenes identified by Methylation PCR arrays. (b) Tumour suppressor genes CDH1 and MGMT were transcriptionally activated by ERp29. The mRNA and protein expressions were examined by RT-PCR and Western blot. (c) MS-PCR analysis for MGMT promoter methylation/demethylation. Note that the ratio of demethylation/methylation was highly increased in the ERp29-transfected cells (clone B and E). Cells treated with 5′ -aza-dC was used as a positive control for demethylation. Genomic DNA was extracted and converted with sodium bisulfite. MS-PCR was performed as described in “Materials and Methods”. **p < 0.01 versus control.

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Positive Control, Control

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 3. ERp29 expression reduces DNMT1 to increase MGMT promoter demethylation in MDA-MB-231 cells. (a) ERp29 expression decreased the level of DNMT1 whereas ERp29 knockdown upregulated the expression of DNMT1. The expression of DNMT3A or 3B was not markedly affected by ERp29. *p < 0.05, **p < 0.01, relative mock-transfected control or controL siRNA. (b) Reduction of DNMT1 by siRNA upregulated MGMT expression in MDA-MB-231 cells. MDA-MB-231 cells were transiently transfected with control siRNA or DNMT1 siRNA (#1) for 48hours and the expression of DNMT1 and MGMT was examined. (c) MGMT promoter methylation/demethylation. Genomic DNA was extracted from the MDA-MB-231 cells transfected with control siRNA or DNMT1 siRNA and the MS-PCR was done as described in “Materials and Methods”. **p < 0.01, ***p < 0.001, versus control.

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Expressing, Knockdown, Transfection, Control, Methylation

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 4. MGMT is a downstream target of ERp29. ERp29-transfected cells (cone B) (a) or MCF7 cells (b) were treated with ERp29 siRNA (#1) or MGMT siRNA (#3) or control siRNA and the expression of ERp29 and MGMT was analysed. ERp29 knockdown decreased the expression of MGMT whereas MGMT knockdown was unable to decrease the level of ERp29 in both clone B cells (a) and MCF-7 cells (b). **p < 0.01, ***p < 0.001, relative to control.

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Transfection, Control, Expressing, Knockdown

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 5. MGMT mediates ERp29-induced post-irradiation survival rate. and facilitates DNA damage in ERp29-transfected MDA-MB-231 cells. (a) Repression of MGMT by siRNA (#3) reduced the ERp29- enhanced post-irradiation survival rate. Cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. Expression of MGMT was efficiently repressed by siRNA in the ERp29-overexpressed clone B cells. (b) Depletion of endogenous MGMT by siRNA in MCF-7 cells sensitized to radiation treatment. MCF-7 cells were treated with control or MGMT siRNA (#3) for 48 hours and then exposed to irradiation at the indicated doses. The expression of MGMT in MCF-7 cells was efficiently reduced by siRNA. (c) Re-expression of MGMT in the MB-231/ERp29 siRNA cells restores radioresistance. Cells were transfected with pcDNA-MGMT or pcDNA for 24 hours and exposed to radiation treatment. MGMT expressed was examined by immunoblot. Post-radiation survival rate was assessed by clonogenic assay as described in Fig. 1

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Irradiation, Transfection, Control, Expressing, Western Blot, Clonogenic Assay

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 6. MGMT repression facilitates DNA damage in ERp29-transfected MDA-MB-231 cells and MCF-7 cells. (a) Reduction of MGMT in ERp29-overexpressed cells increased irradiation-induced expression of γ –H2AX. Note that irradiation induces significant increase of γ –H2AX in the mock- transfected MDA-MB-231 cells (column 2 vs. 1). Depletion of MGMT alone in the ERp29-overexpressed clone B cells only slightly increased the expression of H2AX (column 5 vs. 1). However, combination treatment (column 4) of irradiation/MGMT siRNA in these cells led to a significant induction of γ –H2AX relative to the cells treated with MGMT siRNA (column 5) or with the irradiation/control siRNA (column 3). The level of γ –H2AX was examined after 12 hours of post-irradiation. (b) Irradiation treatment significantly increased the expression of γ –H2AX in MGMT-knockdown MCF-7 cells. MCF-7 cells were transfected with MGMT siRNA or control siRNA for 48 hours and then irradiated with 4 Gy. The expression of γ –H2AX was assayed after 12 hours of post-irradiation. (c) MGMT depletion induced high expression of γ –H2AX and reduced double strand DNA breaks repair after irradiation. MB-231/ERp29 (clone B) cells were treated with MGMT siRNA or control siRNA for 48 hours, irradiated with 4 Gy and incubated for 2, 6, 12 and 24 hours. Phosphorylation of H2AX was examined by Western blot. **p < 0.01, ***p < 0.001, relative to control at the indicated doses/time.

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Transfection, Irradiation, Expressing, Control, Knockdown, Incubation, Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

doi: 10.1038/srep14723

Figure Lengend Snippet: Figure 7. MGMT depletion by siRNA stimulates radiation-induced cell apoptosis in ERp29-transfected MDA-MB-231 cells. (a) Reduction of MGMT in ERp29-overexpressed clone B cells increased irradiation- induced expression of cleaved caspase 3 compared to the control siRNA-treated cells. Expression of cleaved caspase 3 was highly induced by irradiation in the vector-transfected MDA-MB-231 cells (column 2 vs. 1). In the ERp29-overespressed clone B cells, depletion of MGMT alone did not activate expression of cleaved caspase 3 (column 3 vs. 1). However, these cells treated with MGMT siRNA (#3) markedly increased the expression of cleaved caspase 3 after irradiation (column 5 vs. 4). (b) Cell viability. Irradiation treatment caused significant reduction of cell viability in vector-transfected MDA-MB-231 cells (column 2 vs. 1). Combinatory treatment of irradiation/MGMT siRNA (column 5) in the ERp29-overexpressed clone B cells significantly reduced cell viability compared to the cells treated with MGMT siRNA alone (column 3) or the irradiation/control siRNA (column 4). **p < 0.01, ***p < 0.001, relative to control.

Article Snippet: 1 1Scientific RepoRts | 5:14723 | DOi: 10.1038/srep14723 For construction of MGMT expression vector, MGMT human cDNA (Origene) were amplified using forward primer 5′ -CGGGATCCGG CACCATGGAC AAGGATTGTG AAATGAAACG-3′ with BamHI; and reverse primer 5′ -CTCTAGATCA GTTTCGGC CAGCAGGCGG GGAGC-3′ with XbaI.

Techniques: Transfection, Irradiation, Expressing, Control, Plasmid Preparation

Journal: Clinical Cancer Research

Article Title: Profound Prevention of Experimental Brain Metastases of Breast Cancer by Temozolomide in an MGMT-Dependent Manner

doi: 10.1158/1078-0432.ccr-13-2588

Figure Lengend Snippet: Figure 4. MGMT expression in matched sets of human breast primary tumors and resected brain metastases. A to D, sixty-two patient-matched sets were collected from tumor banks in Poland and Germany. TMAs of the specimens were stained for MGMT and evaluated for the percentage of positively staining tumor cells (nuclear staining only), dichotomized at 5%. The number and percentage of specimens in each category is given below each representative photomicrograph.

Article Snippet: Briefly, the human MGMT cDNA was purchased from Origene Technologies and cloned into the pCDH-CMVHygro lentiviral vector (Systems BioScience) for lentivirus production and subsequent infection of cells per the manufacturer’s recommended protocol.

Techniques: Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: O 6 -methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo , and sensitivity to alisertib-carboplatin combination treatment

doi: 10.3389/fncel.2025.1552015

Figure Lengend Snippet: Characterization of U1210 O 6 -methylguanine DNA methyltransferase (MGMT) knockout (KO) cells. (A) Confirmation of MGMT KO in U1242 glioblastoma (GBM) cells by western blotting. (B) MGMT KO confers temozolomide (TMZ) sensitivity to U1242 cells. Wildtype U1242 cells are resistant to TMZ induced apoptosis as indicated by minimal detection of cleaved PARP (C PARP). MGMT KO U1242 cells undergo substantially more apoptosis after TMZ treatment. Cells were treated with 20 μM TMZ for 72 h and subjected to western blotting. (C) Wildtype and MGMT KO U1242 cells exhibit nearly identical proliferation rates. Growth curves of MGMT WT and MGMT KO cells were performed in vitro .

Article Snippet: Two separate guide RNAs (gRNAs) that target exon 2 of the MGMT gene are provided by the Origene MGMT Knockout Kit ( KN201612 ).

Techniques: Knock-Out, Western Blot, In Vitro

Journal: Frontiers in Cellular Neuroscience

Article Title: O 6 -methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo , and sensitivity to alisertib-carboplatin combination treatment

doi: 10.3389/fncel.2025.1552015

Figure Lengend Snippet: The effect of O 6 -methylguanine DNA methyltransferase (MGMT) expression on 3D growth of U1242 cells. (A) Representative photomicrographs of U1242 MGMT wildtype (WT) and knockout (KO) cell growth in soft agar on days 1, 4, 7, and 10 (scale bars represent 50 μm). (B) Representative photomicrographs of U1242 MGMT WT cells, transfected with mock or MGMT siRNA, in soft agar on days 1, 4, 7, and 10 (scale bars represent 50 μm). (C) Quantification of colony sizes of MGMT WT and KO cells. (D) Quantification of mock or MGMT siRNA colony sizes transfected U1242 MGMT WT cells. (E) Confirmation of MGMT knockdown on days 1 and 4 in U1242 cells.

Article Snippet: Two separate guide RNAs (gRNAs) that target exon 2 of the MGMT gene are provided by the Origene MGMT Knockout Kit ( KN201612 ).

Techniques: Expressing, Knock-Out, Transfection, Knockdown

Journal: Frontiers in Cellular Neuroscience

Article Title: O 6 -methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo , and sensitivity to alisertib-carboplatin combination treatment

doi: 10.3389/fncel.2025.1552015

Figure Lengend Snippet: Comparison of in vivo growth of U1242 O 6 -methylguanine DNA methyltransferase (MGMT) wildtype (WT) and knockout (KO) cells. (A) Cell lines and implanted cell numbers for each experimental group. (B) Representative H&E staining of mouse brains; (i): MGMT WT 20X, (ii): MGMT WT 50X, (iii): MGMT KO 20X, (iv): MGMT KO 50X ( n = 3 for each group, scale bars represent 200 μm).

Article Snippet: Two separate guide RNAs (gRNAs) that target exon 2 of the MGMT gene are provided by the Origene MGMT Knockout Kit ( KN201612 ).

Techniques: Comparison, In Vivo, Knock-Out, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: O 6 -methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo , and sensitivity to alisertib-carboplatin combination treatment

doi: 10.3389/fncel.2025.1552015

Figure Lengend Snippet: Effect of alisertib and/or carboplatin treatment on (A) O 6 -methylguanine DNA methyltransferase (MGMT) wildtype (WT) and knockout (KO) cells, and (B) Empty or MGMT overexpression vector transfected MGMT KO cell line (Each data point represents the mean value of three replicates from an independent experiment, n = 3, two-way ANOVA). (C) Kaplan-Meier survival curve of orthotopic xenograft U1242 MGMT WT cells implanted mice (vehicle, n = 5; alisertib only, n = 7; carboplatin only, n = 6; and alisertib + carboplatin, n = 7).

Article Snippet: Two separate guide RNAs (gRNAs) that target exon 2 of the MGMT gene are provided by the Origene MGMT Knockout Kit ( KN201612 ).

Techniques: Knock-Out, Over Expression, Plasmid Preparation, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: O 6 -methylguanine DNA methyltransferase (MGMT) expression in U1242 glioblastoma cells enhances in vitro clonogenicity, tumor implantation in vivo , and sensitivity to alisertib-carboplatin combination treatment

doi: 10.3389/fncel.2025.1552015

Figure Lengend Snippet: Changes in DNA damage markers in U1242 O 6 -methylguanine DNA methyltransferase (MGMT) wildtype (WT) and knockout (KO) cells with alisertib and carboplatin treatment. (A) Representative western blots of DNA damage response markers in MGMT WT and MGMT KO cells treated with 0 to 10 μM alisertib, carboplatin, or alisertib + carboplatin. Quantification of (B) pATM, (C) pBRCA1, (D) pChk1, (E) . pChk2, and (F) pHistone-H2AX protein levels. Each data point represents the values from independent experiments. n = 3, two-way ANOVA.

Article Snippet: Two separate guide RNAs (gRNAs) that target exon 2 of the MGMT gene are provided by the Origene MGMT Knockout Kit ( KN201612 ).

Techniques: Knock-Out, Western Blot

Journal: PLoS ONE

Article Title: Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response

doi: 10.1371/journal.pone.0152684

Figure Lengend Snippet: Fluorescence spectra showing the overall fold-changes in intensity observed when comparing fluorescence measured before (dashed line) and after (solid line) addition of purified MGMT protein are shown at left of each figure. Time courses (on the right) show time-dependent fluorescence increases immediately after addition of enzyme. Final probe and MGMT concentrations were 100 nM. Assays were run at 37°C in 70 mM HEPES buffer pH 7.8 containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. (A) chemosensor 1 containing dT FAM , (B), chemosensor 2 containing Cy3, (C), chemosensor 3 containing dT TMR and (D), chemosensor 4 containing perylene nucleoside. Measurements were repeated 3 times. Standard deviations are provided in .

Article Snippet: Purified recombinant human MGMT (His-tagged, expressed in E . Coli ) was purchased from Creative BioMart.

Techniques: Fluorescence, Purification

Journal: PLoS ONE

Article Title: Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response

doi: 10.1371/journal.pone.0152684

Figure Lengend Snippet: Incubation of purified MGMT enzyme with the inhibitors BG and PaTrin-2 led to a concentration dependent decrease in observed final fluorescence intensity, indicative of MGMT inhibition. MGMT (10 nM) was incubated with inhibitor for 10 min at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. Final fluorescence was acquired 10 min after addition of probe (10 nM). Data were normalized to measurements without inhibitor. Each data point is the average of 3 measurements.

Article Snippet: Purified recombinant human MGMT (His-tagged, expressed in E . Coli ) was purchased from Creative BioMart.

Techniques: Incubation, Purification, Concentration Assay, Fluorescence, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Biochemical reconstitution of temozolomide-induced mutational processes

doi: 10.1016/j.jbc.2025.110676

Figure Lengend Snippet: Influence of TLS polymerases on the TMZ-induced mutation spectra in the presence and absence of hMGMT. A, illustration of the experimental system. dsDNA substrates were incubated with 400 μM TMZ three times. When indicated, damaged templates were treated with hMGMT. The DNA was heated and reannealed with NGS primer and 10x excess competitor to sequester the top strand. Then the primer extension was started by adding yPol δ. After 30 min of incubation with yPol δ, the second polymerase (either yPol ζ, hPol κ, or hPol η) was added, and the reaction was continued for another 30 min. B – D, mutation spectra produced on the TMZ-damaged DNA in the presence of the indicated second polymerase without hMGMT treatment. E–G, influences of the second polymerase on the C>T mutations were expressed as a ratio of the mutation frequencies at individual sites. CpC>T and CpT>T mutations (SBS11), other mutations (Others), and all mutations (All) are plotted as separate groups. H – J, the same experiments as in B – D were carried out using the templates that were treated with hMGMT. K, influences of hMGMT on the C>T and C>A mutations that were produced in the presence of indicated second polymerases. For hPol η reactions, only C>T mutations were analyzed because this polymerase did not produce considerable C>A mutations. Mutation frequencies mapped on the templates are shown in . hMGMT, human methylguanine methyltransferase; hPol κ, human Pol κ; hPol η, human Pol η; NGS, next-generation sequencing; SBS11, substitution signature 11; TLS, translesion synthesis; TMZ, temozolomide; yPol δ, yeast Pol δ.

Article Snippet: Human complementary DNA of MGMT (hMGMT) was obtained from SinoBiological , amplified by PCR, and cloned into pET21a to express MGMT with a C-terminal His6-tag.

Techniques: Mutagenesis, Incubation, Produced, Next-Generation Sequencing, Translesion Synthesis