Journal: PLoS ONE

Article Title: Developing a clinically relevant radiosensitizer for temozolomide-resistant gliomas

doi: 10.1371/journal.pone.0238238

Figure Lengend Snippet: MGMT-expressing variants (i.e., LN229M, U251M, and GL261M) of three well-established GB cell lines were checked along with their parental cells as well as the T98G (an endogenous high MGMT expressor) and LN229TR2 (an MMR-deficient variant of LN229) GB cells for MGMT expression by Western blotting (panel A). Colony survival data are also shown for all three isogenic GB cell lines (panel B). The O6BG MGMT inhibitor (40 μΜ) was used to demonstrate that the artificially expressed MGMT proteins in LN229M, U251M, and GL261M cells were functional and respond to specific inhibition (i.e., the MGMT-positive cells are sensitized to both alkylating agents after the addition of the O6BG inhibitor).

Article Snippet: A rabbit polyclonal antibody that crossreacts with both human MGMT and mouse Mgmt proteins was purchased from Boster Biological Technology (Pleasanton, CA) and used in this Western-blot analysis.

Techniques: Expressing, Variant Assay, Western Blot, Functional Assay, Inhibition

Journal: Pharmaceutics

Article Title: Therapeutic Options for Recurrent Glioblastoma—Efficacy of Talaporfin Sodium Mediated Photodynamic Therapy

doi: 10.3390/pharmaceutics14020353

Figure Lengend Snippet: Patient demographics and characteristics.

Article Snippet: The methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) was not evaluated, but alternatively, the expression of MGMT protein was determined by conducting immunohistochemistry with anti-MGMT monoclonal antibody (MAB16200, Merck, Darmstadt, Germany).

Techniques: Control, Histopathology, Mutagenesis, Expressing

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: MGMT mRNA was quantified in FineFix–fixed and paraffin-embedded 57 samples of glioblastoma (WHO grade IV). MGMT gene promoter was non-methylated in all samples. MGMT mRNA relative expression normalized over GAPDH mRNA was analyzed by RT-qPCR with TaqMan probes. ( A ) Relative expression was ordered in a rank in order to select samples at the extremes of low (L) and high (H) MGMT mRNA expression. ( B ) Expression pattern of five miRNAs differentially expressed between MGMT-low and MGMT-high glioblastoma samples (WHO grade IV), displaying extreme levels of MGMT mRNA. MGMT gene promoter was non-methylated in all samples. ( C ) MiR-181 family expression in the same low- and high-MGMT glioblastoma samples.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Methylation, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: ( A ) Quantitative analyses of miR-127-3p, ( B ) miR-409-3p, ( C ) miR-129-5p, ( D ) miR-124-3p, ( E ) miR-381-3p, ( F ) miR-181d-5p, ( G ) miR-181c-3p, ( H ) miR-181c-5p and ( I ) miR-181a-5p. The two groups of extreme samples refer the specimens expressing to low ( n = 5) and high ( n = 5) MGMT mRNA. Analyses were performed with TaqMan probes and each miRNA was normalized to the most stable miRNA of the array (miR-760). Mean (bold line), median (light line), 5th and 95th confidence interval (box limits) and outliers as single dots are reported.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Expressing

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: ( A ) miR-127-3p, ( B ) -409-3p, ( C ) -124-3p, ( D ) -181d-5p, ( E ) -648, ( F ) -767-3p and ( G ) -603 in WHO 2007 grade IV glioblastoma with non-methylated MGMT promoter ( n = 26).

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Methylation

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: T98G glioblastoma cells were transfected with 200 nM of the pre-miRs indicated in the figure labels for each microRNA or scrambled sequence (−). ( A ) MGMT mRNA expression was detected with TaqMan probes, as indicated in the Method section, and the relative expression normalized to that of T98G cells transfected with scrambled sequence. ( B ) MGMT protein expression as detected by Western analysis is shown in the inset panel (using β-actin as reference). Band density was plotted and normalized to that of the T98G cells transfected with the scrambled sequence. Statistical significance in A and B at the levels of p < 0.05 (*) and p < 0.01 (**) is relative to cells transfected with scrambled sequence.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Transfection, Sequencing, Expressing, Western Blot

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: MGMT expression is increased by different nuclear transcription factors (Sp1, NF-kB, AP-1, CEBP and HIF-1a), together with the acetylation of histones H3 and H4 and the stabilization by binding of N-myc Downstream Regulated Gene 1 (NDRG1) protein (for review see ). On the opposite, MGMT expression is downregulated by different mechanisms, namely methylation of the CpG islands in the promoter , di-methylation of histone H3K9 and microRNAs. The graph represents the central role of miR-181d-5p in down regulating MGMT expression, by acting both on MGMT mRNA degradation and interference with protein translation. Other and this report suggest that miR-603 , and 409-3p cooperate with miR-181d-5p in MGMT mRNA degradation, whereas miR-648 , miR-767-3p , -124-3p and 127-3p could play an ancillary role.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Expressing, Binding Assay, Methylation