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

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