Journal: Oncology Letters

Article Title: NF-κB inhibitor reverses temozolomide resistance in human glioma TR/U251 cells

doi: 10.3892/ol.2015.3130

Figure Lengend Snippet: Expression of MGMT, Bcl-2 and p65 in the BAY 11-7082, TMZ and combination treatment groups. A high expression of MGMT was found in the TR/U251 cells, while expression was hardly detected in the U251 cells. Similarly, the levels of phosphorylated-P65 were increased in the TR/U251 cells compared with the U251 cells. No statistical difference was found in the NF-κB expression, assessed through the detection of the p65 subunit, between the two cell types. The TR/U251 cells demonstrated a ~5.4-fold higher level of phosphorylated-P65 compared with the U251 cells. MGMT was hardly detected when cells were cultured with a combination of TMZ and BAY 11-7082. A higher level of MGMT was detected in the cells treated with TMZ or BAY 11-7082 separately. The expression of Bcl-2 and Bax was reversed only in the cells treated with a combination of TMZ and BAY 11-7082. TMZ, temozolomide; TR, TMZ-resistant; MGMT, O6-methylguanine-DNA methyltransferase; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; p-p65, phosphorylated p65.

Article Snippet: All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), consisting of primary mouse anti-human polyclonal antibodies against MGMT (cat. no. sc-400720-HDR), NF-κB (p65; cat. no. sc-8008 AC) and β-actin (cat. no. sc-77326-PR), rabbit anti-human polyclonal antibodies against phosphorylated p65 (cat. no. sc-45101), B-cell lymphoma (Bcl)-2 (cat. no. sc-130916) and Bcl-2-associated X protein (cat. no. sc-377576), and secondary HRP-conjugated rabbit anti-goat (cat. no. sc-2768) antibodies.

Techniques: Expressing, Cell Culture

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: American Journal of Translational Research

Article Title: Differentiated embryonic chondrocyte-expressed gene 1 promotes temozolomide resistance by modulating the SP1-MGMT axis in glioblastoma

doi:

Figure Lengend Snippet: DEC1 promotes TMZ resistance in MGMT-positive GBM cells. Stable GBM cell lines (T98G, LN18, and U251) were established by an infection with DEC1- or shDEC1-recombinant lentivirus and selection with puromycin. (A and B) LN18 and T98G were treated with 600 μM TMZ at different time points. cell survival was evaluated with CCK-8 assays (n = 3). (C and D) Cells were treated with TMZ (600 μM for LN18 and T98G, and 200 μM for U251) for 48 hours; cell apoptosis was evaluated using flow cytometry analysis. Representative images are shown (n = 3). (E) Cells were treated with 600 μM TMZ for 48 hours; the expression level of DEC1 and Bcl-2 were evaluated using Western blot (n = 3). In (A-D), data are presented as the mean ± SD. Unpaired t test for two-groups datasets. *; P < 0.05, **; P < 0.01, ***; P < 0.001. CCK-8, cell counting kit 8; DEC1, differentiated embryonic chondrocyte; GBM, glioblastoma multiforme; MGMT, methylguanine methyltransferase; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SD, standard deviation; shDEC1, short hairpin differentiated embryonic chondrocyte; specificity protein 1; TMZ, temozolomide.

Article Snippet: Lentivirus plasmids containing full-length DEC1 (EX-DEC1-pEZ-Lv105) or MGMT (EX-MGMT-pEZ-Lv151) or their corresponding empty cherry plasmids (i.e., EX-pEZ-Lv105 and EX-pEZ-Lv151) were commercially purchased (Guangzhou Fulengen Co., Ltd., Guangdong, China).

Techniques: Infection, Recombinant, Selection, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Cell Counting, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: American Journal of Translational Research

Article Title: Differentiated embryonic chondrocyte-expressed gene 1 promotes temozolomide resistance by modulating the SP1-MGMT axis in glioblastoma

doi:

Figure Lengend Snippet: DEC1 positively regulates SP1 and MGMT. (A) The expression level of DEC1, MGMT and GAPDH in stable GBM cells was detected with Western blot analysis (n = 3). (B and C) The transcriptional levels of MGMT was evaluated using RT-qPCR (n = 3). (D) The 293T cells were transiently co-transfected with the DEC1-encoding construct or empty vector, and the control luciferase vector pGL3.0-Luc or the luciferase reporter pGL3.0-pMGMT-Luc. Luciferase activity was evaluated 48 hours after transfection and shown as the ratio of promoter-reporter activity to control vector luciferase activity (n = 3). (E) The LN18 cells were transfected with siNC, siSP1#1, siSP1#2 or siSP1#3. The expression levels of SP1, DEC1, and MGMT were evaluated with Western blot (n = 3). (F) In stable DEC1 GBM cell lines (i.e., T98G, LN18, and U251), the expression level of SP1 along with DEC1 and MGMT were evaluated with Western blot (n = 3). (G) Stable DEC1-overexpressing LN18 was transiently transfected with siSP1#1 or siNC. The expression levels of MGMT along with SP1 and DEC1 were evaluated with Western blot. In (B-D), the data are presented as the mean ± SD. Unpaired t test for two-groups datasets. *; P < 0.05, **; P < 0.01, ***; P < 0.001.

Article Snippet: Lentivirus plasmids containing full-length DEC1 (EX-DEC1-pEZ-Lv105) or MGMT (EX-MGMT-pEZ-Lv151) or their corresponding empty cherry plasmids (i.e., EX-pEZ-Lv105 and EX-pEZ-Lv151) were commercially purchased (Guangzhou Fulengen Co., Ltd., Guangdong, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Control, Luciferase, Activity Assay

Journal: American Journal of Translational Research

Article Title: Differentiated embryonic chondrocyte-expressed gene 1 promotes temozolomide resistance by modulating the SP1-MGMT axis in glioblastoma

doi:

Figure Lengend Snippet: The SP1-MGMT axis is essential for DEC1-mediated TMZ resistance. After being individually infected with DEC1 or shDEC1 recombinant lentivirus, the T98G and LN18 cells were further infected with MGMT, shMGMT or their individual empty control recombinant lentivirus and selected with hygromycin and G418 to establish stable cell lines. (A) Cells were treated with TMZ (600 μM) for 48 hours. The expression level of MGMT, DEC1, and Bcl-2 were determined by Western blot. (B) Cells indicated were treated with different concentration of TMZ for 48 hours, and cell survival was evaluated by CCK-8 assay (n = 3). (C and D) Cells were treated with 600 μM TMZ for 48 hours and cell apoptosis was evaluated by flow cytometry analysis (n = 3). Representative images are shown in (C). In (B and D), data are presented as the mean ± the SD. Unpaired t test for two-groups datasets. *; P < 0.05, **; P < 0.01, ***; P < 0.001, ****; P < 0.0001.

Article Snippet: Lentivirus plasmids containing full-length DEC1 (EX-DEC1-pEZ-Lv105) or MGMT (EX-MGMT-pEZ-Lv151) or their corresponding empty cherry plasmids (i.e., EX-pEZ-Lv105 and EX-pEZ-Lv151) were commercially purchased (Guangzhou Fulengen Co., Ltd., Guangdong, China).

Techniques: Infection, Recombinant, Control, Stable Transfection, Expressing, Western Blot, Concentration Assay, CCK-8 Assay, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Differentiated embryonic chondrocyte-expressed gene 1 promotes temozolomide resistance by modulating the SP1-MGMT axis in glioblastoma

doi:

Figure Lengend Snippet: SP1 regulates TMZ sensitivity via MGMT. The T98G and LN18 cells infected with MGMT or empty Cherry recombinant lentivirus were further transiently transfected with siSP1#NC or siSP1#1. A. Cells were treated with TMZ at 600 μM for 48 hours. The expression level of SP1, MGMT, DEC1, and Bcl-2 were analyzed using Western blot. B. Cells were treated with different concentrations of TMZ for 48 hours and cell survival were evaluated using CCK-8 assays (n = 3). C. Cell apoptosis was evaluated by using flow cytometry analysis (n = 3). Data are presented as the mean ± the SD. Unpaired t test for two-groups datasets. *; P < 0.05, **; P < 0.01, ***; P < 0.001, ****; P < 0.0001.

Article Snippet: Lentivirus plasmids containing full-length DEC1 (EX-DEC1-pEZ-Lv105) or MGMT (EX-MGMT-pEZ-Lv151) or their corresponding empty cherry plasmids (i.e., EX-pEZ-Lv105 and EX-pEZ-Lv151) were commercially purchased (Guangzhou Fulengen Co., Ltd., Guangdong, China).

Techniques: Infection, Recombinant, Transfection, Expressing, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Differentiated embryonic chondrocyte-expressed gene 1 promotes temozolomide resistance by modulating the SP1-MGMT axis in glioblastoma

doi:

Figure Lengend Snippet: The expression of DEC1 is upregulated in GBM and correlated with the expression of MGMT. A. The expression level of DEC1 was analyzed using Western blot in normal human brain cell line HEB and GBM cell lines (T98G, LN18, and U251). B. The correlation between the expression level of DEC1 and the glioma grade was analyzed, based on the database information provided by the Cancer Genome Atlas (TCGA). C and D. The expression level of DEC1 and MGMT were analyzed by immunohistochemical staining of glioma tissue microarray, which contains 35 GBM tissues and 5 normal brain tissues. E. Representative immunohistochemistry images with high and low levels of DEC1 and MGMT are shown. F. The IRS scores of DEC1 and that of MGMT (low expression group: IRS < 6 or high expression group: IRS ≥ 6) were counted and the percentage of high/low MGMT in DEC1 high/low were shown. P < 0.05, chi-square test was applied.

Article Snippet: Lentivirus plasmids containing full-length DEC1 (EX-DEC1-pEZ-Lv105) or MGMT (EX-MGMT-pEZ-Lv151) or their corresponding empty cherry plasmids (i.e., EX-pEZ-Lv105 and EX-pEZ-Lv151) were commercially purchased (Guangzhou Fulengen Co., Ltd., Guangdong, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Microarray, Immunohistochemistry