Journal: bioRxiv

Article Title: A quantitative characterization of the heterogeneous response of glioblastoma U-87 MG cell line to temozolomide

doi: 10.1101/2024.05.28.596108

Figure Lengend Snippet: (A) lifespan versus mean fluorescence of MGMT-C-GFPspark, for single cells from the control (green) and treated (red) populations. (B) As in (A) but for mean speed versus mean fluorescence.

Article Snippet: Eukaryotic expression plasmid for human MGMT tagged with GFPspark was purchased from SinoBiological (HG12077-ACG).

Techniques: Fluorescence

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

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: Drug‐resistant lung cancer cells increased expression of Trps1 and MGMT . (A) The morphology of established H446/ CDDP cells. Scale bars: 200 mm. (B) Cells growth curve of H446/ CDDP and parental H446 cell. (C) Semi‐ QPCR and Western blot analyze the expression of Trps1 and MGMT in H446 and H446/ CDDP cells. * P < 0.05 versus H446 control; (D) immunofluorescence assay the expression of Trps1 and MGMT in H446 and H446/ CDDP cells. Scale bars: 50 mm. All figures represent the average of three sets of independent experiments.

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Expressing, Western Blot, Control, Immunofluorescence

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: Effects of Trps1 expression on MGMT mRNA and protein levels. (A) The strategy of overexpression Trps1 using plasmid pL enti‐ CMV ‐ GFP ‐Puro. (B) QPCR assay the effect of alternation Trps1 expression on the mRNA level of indicated genes. (C) Western blot analyzes the effect of alternation Trps1 expression on the protein level of indicated genes. * P < 0.05 versus H446 and H446‐Le/ GFP control groups; ▲, P < 0.05 versus H446/ CDDP and H446/ CDDP ‐ NS si RNA control groups. All figures represent the average of three sets of independent experiments.

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

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

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: Effects of MGMT expression on multidrug sensitivity of lung cancer cells. MGMT and Trps1 gene expressions were analyzed at mRNA (A) and protein (B) levels in MGMT stably overexpressed H446 cells. * P < 0.05 versus H446 and H446‐Le/ GFP control groups; (C) the IC 50 value of cisplatin in MGMT overexpressed H446 cells. (D) The IC 50 value of 5‐ FU in MGMT overexpressed H446 cells. * P < 0.05 versus H446 and H446‐Le/ GFP control groups. All figures represent the average of three sets of independent experiments.

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Expressing, Stable Transfection, Control

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: MGMT expression partly reverses the effect of silencing Trps1 on multidrug sensitivity of lung cancer cells. MGMT and Trps1 gene expressions were analyzed at mRNA (A) and protein (B) levels in MGMT stably overexpressed and Trps1 silenced H446/ CDDP cells. * P < 0.05 versus H446/ CDDP ‐ NS si RNA control. (C) The IC 50 value of cisplatin in MGMT overexpressed and Trps1 silenced H446/ CDDP cells. (D) The IC 50 value of 5‐ FU in MGMT overexpressed and Trps1 silenced H446/ CDDP cells. ▲, P < 0.05 versus H446/ CDDP ‐ NS si RNA control group; #, P < 0.05 versus H446/ CDDP ‐Trps1 si RNA group. (E) MGMT and Trps1 gene expressions were analyzed at protein level in Trps1 stably overexpressed and MGMT transiently silenced H446 cells. * P < 0.05 versus H446‐Le/Trps1 cells. The IC 50 value of cisplatin (F) and 5‐ FU (G) in Trps1 overexpressed and MGMT silenced H446 cells. * P < 0.05 versus H446‐Le/Trps1 cells. All figures represent the average of three sets of independent experiments.

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Expressing, Stable Transfection, Control

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: Trps1 transcriptional regulates MGMT expression by binding to the MGMT promoter. (A) Schematic representation of two Trps1 binding sites in the MGMT promoter. (B) H446 cells and H446‐Le/Trps1 cells were transfected with MGMT reporter plasmid, and the luciferase activity was measured. * P < 0.05 versus H446 group. (C) H446/ CDDP cells were cotransfected with MGMT reporter plasmid and anti‐ TRPS 1 si RNA , and the luciferase activity was measured. ▲, P < 0.05 versus H446/ CDDP ‐ NS si RNA group. (D) H446‐Le/Trps1 cells were transfected indicated MGMT WT or mutated reporter plasmids, and the luciferase activity was measured. #, P < 0.05 versus WT group. (E) Ch IP ‐ PCR assay for the Trps1 binding sites in the promoter of the MGMT gene. All figures represent the average of three sets of independent experiments.

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: The oligonucleotide primers used in plasmids construction

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Sequencing

Journal: Cancer Medicine

Article Title: Trps1 is associated with the multidrug resistance of lung cancer cell by regulating MGMT gene expression

doi: 10.1002/cam4.1421

Figure Lengend Snippet: The oligonucleotide primers used in RT and ChIP‐PCR assay

Article Snippet: H446‐Le/Trps1 cells were transiently transfected with human MGMT short interfering RNA mix (siRNAm) (Genepharma, Pudong, Shanghai, China).

Techniques: Sequencing