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: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: MGMT expression levels in GBM, GSCs and melanoma cell lines. ( A and D ) The methylation-specific PCR (MSP) analyses of the MGMT promoter from GBMs, GSCs, and melanoma cell lines. Note the bands in the unmethylated (U, 93 bp) and methylated (M, 81 bp) lanes for GBM, GSCs, and melanoma cell lines, reflecting the unmethylated/methylated MGMT promoter. Percentages represent the proportion of methylation (M) and unmethylation (U) on the MGMT promoter in each cell line. ( B and E) Transcript levels of MGMT mRNA in GBM, GSCs, and melanoma cell lines were determined by qRT-PCR. ( C and F) MGMT levels in GBM, GSCs, and melanoma cell lines were determined by immunoblot analysis. Data are the mean ± SEM for three independent experiments.

Article Snippet: Human MGMT cDNA (MGMT [untagged]-human O 6 -methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors.

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of siMGMT on the radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with or without 25 nM siMGMT for 48 h. MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were transfected with 25 nM of si (negative control) and siMGMT before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by siMGMT alone. Data are the mean ± SEM for three independent experiments. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: Human MGMT cDNA (MGMT [untagged]-human O 6 -methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors.

Techniques: Transfection, Western Blot, Negative Control, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radioresponse of MGMT-producing cells. ( A and B ) ACPK1, GBMJ1, A375, and MM415 cells were treated with a gradient (25 µM, 50 µM, 100 µM, and 150 µM) of lomeguatrib for the indicated times (24 h and 48 h). MGMT levels were determined by immunoblot analysis. ( C and D ) ACPK1, GBMJ1, A375, and MM415 cells were treated with the designated concentrations of lomeguatrib (ACPK1, A375, and MM415—100 µM and GBMJ1—50 µM) for 16 h before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by lomeguatrib alone. * P < 0.05 and ** P < 0.005 by Student’s t-test.

Article Snippet: Human MGMT cDNA (MGMT [untagged]-human O 6 -methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors.

Techniques: Western Blot, Generated

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of lomeguatrib on radiation-induced γH2AX foci and mitotic catastrophe in MGMT-producing cells. ( A ) The quantitative assessment of γH2AX foci per cell at 1 h and 24 h after radiation is shown. Foci were counted in at least 50 cells per experiment and representative histograph images obtained from control, lomeguatrib-only, radiation (4 Gy)-only, and lomeguatrib/radiation combination treatment. Data are the mean ± SEM for three independent experiments, and statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.005 (radiation versus lomeguatrib/radiation). ( B ) The quantitative assessment of nuclear fragmentation per cell at 24 h and 72 h after radiation is shown. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was evaluated in at least 100 cells per treatment per experiment and representative histograph images obtained from control cells and cells pretreated with lomeguatrib alone, radiation (4 Gy) alone, and combination treatment of lomeguatrib with radiation. Data are the mean ± SEM for three to four independent experiments. Statistical significance was determined by Student’s t-test. *P < 0.05 and ***P < 0.0005 (radiation versus Lomeguatrib + radiation).

Article Snippet: Human MGMT cDNA (MGMT [untagged]-human O 6 -methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors.

Techniques: Control

Journal: Scientific Reports

Article Title: MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma

doi: 10.1038/s41598-024-61240-x

Figure Lengend Snippet: The effect of MGMT overexpression on the radioresponse in non-MGMT-producing cells. ( A ) MGMT protein expression was assessed via western blot analysis following transfection of 1 µg GFP-MGMT vector in non-MGMT-producing cells (OSU61, NSC11, WM852 and WM266-4) for 48 h. ( B and C ) OSU61, NSC11, WM852 and WM266-4 cells were transfected with 1 µg of GFP-control vector and GFP-MGMT vector before radiation. Surviving fraction (Log) curves were generated after normalizing for the cytotoxicity generated by GFP- MGMT vector alone. DMF values were calculated at a surviving fraction (Log) of 0.1. Data are the mean ± SEM for three independent experiments. * P < 0.05 by Student’s t-test.

Article Snippet: Human MGMT cDNA (MGMT [untagged]-human O 6 -methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Generated