Journal: Scientific Reports

Article Title: DNA methylation in demyelinated multiple sclerosis hippocampus

doi: 10.1038/s41598-017-08623-5

Figure Lengend Snippet: Demyelination in MS brains leads to lower expression of TET enzyme mRNA and hydroxymethyl residues. RT-PCR analysis shows significant decreases in mRNA levels of TET 1, TET 2, and TET 3 in demyelinated hippocampus (n = 4) compared to myelinated hippocampus (n = 4) ( A ). Compared to myelinated hippocampus (n = 8), demyelination (n = 7) led to a significant decrease in levels of hydroxymethyl content (hmC) ( B ). Immunohistochemistry analysis using a 5hmC antibody shows neurons as the major cell type with 5 hmC expression in myelinated ( C ) and demyelinated hippocampus ( D ). Scale Bars: C–D: 30 μm; Error bars indicate + S.E.M.; *p < 0.05.

Article Snippet: Isolated DNA (100 ng) was used to measure DNA hydroxymethylation with MethylFlashTM Global DNA Hydroxymethylation (5hmC) ELISAs (Epigentek Inc, USA) using a 5hmC mAb-based detection complex.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool

doi: 10.1016/j.omtn.2020.08.010

Figure Lengend Snippet: The m6A Methyltransferase METTL3, the m6A Demethylase FTO, and α-Ketoglutarate Regulate the N6-Adenosine Methylation of miR-200b-3p (A) Each bar represents the relative expression level of miR-200b-3p (miR-200b-3p exp ) in 32 samples of GBM. After miRNA extraction from tumors, qRT-PCR was performed to evaluate the relative expression level of miRNA-200b-3p by using non-tumor brain samples as a reference and SNORD6.1 as a housekeeping miRNA. (B) Correlation between the relative expression level of miR-200b-3p and the percentage of N6-adenosine methylation in miR-200b-3p (miR200b-3p %m6A ). This percentage was calculated via the realization of RNA immunoprecipitation (IP) performed with an anti-m6A antibody followed by qPCR analysis (miRIP m6A -qPCR) (as previously described by Berulava et al. ). Each square represents a GBM patient. (C) Absence of correlation between the relative expression level of FTO and the percentage of N6-adenosine methylation in miR-200b-3b. A human FTO ELISA kit (Tebu-Bio, France) was used to estimate the FTO expression. Each square represents a GBM patient. (D) Absence of correlation between the relative presence of α-ketoglutarate (αKG) and the percentage of N6-adenosine methylation in miR-200b-3p. An αKG assay kit (Abcam, France) was used to estimate the relative presence of αKG. Each square represents a GBM patient. (E) High percentage of N6-adenosine methylation in miR-200b-3p is observed in GBM harboring a low level of FTO and αKG. Each square represents a GBM patient. Red squares represent the average ± standard deviation of the two considered subgroups. (F) Impact of the downregulation of FTO on the adenosine methylation percentage of miR-200b-3p% m6A calculated through the realization of miRIP m6A -qPCR as previously described. (G) Impact of the αKG treatment on the adenosine methylation percentage of miR-200b-3p (miRNA-200b-3p %m6A ). (H) Impact of the meclofenamic acid (MA) treatment on miRNA-200b-3p %m6A in U87 cells. After MA treatment (15 μM/24 h, Santa Cruz, France), the miRNA-200b-3p %m6A was calculated through the realization of miRIP m6A -qPCR as previously described. (I) Correlation between the relative expression level of METTL3 and the percentage of N6-adenosine methylation in miR-200b-3p. Each square represents a GBM patient. (J) Dot blot illustrating the presence of adenosine methylation in mimetic miR-200b-3p in the presence of METTL3 IP product. Adenosine detection is used as a control. S1/S2/S3 are three independent experiments. Ctrl, synthetic miR-200b-3p adenosine unmethylated; IP-IgG, product of IP performed with anti-IgG (the absence of an adenosine signal indicates that miRNA was not unspecifically immunoprecipitated); IP-METTL3, product of IP performed with anti-IgG. Products of IP were obtained in non-denaturing conditions in order to conserve enzymatic activity according to the manufacturer’s indications. (K) METTL3 knockdown (by siRNA approach) decreases the percentage of adenosine methylation of miR-200b-3p. (L) αFM score reflects the METTL3, FTO, and αKG expression levels in the 32 GBM samples. Each circle represents a GBM patient. The αFM score is higher when the percentage of adenosine methylation of miR-200b-3p is higher.

Article Snippet: An XIAP (human) cell-based ELISA kit (Abnova, Taiwan), αKG assay kit (no. ab83431, Abcam, France), human FTO ELISA kit (no. 68ELH-FTO, Tebu-Bio, France), METTL3 ELISA kit (no. MBS9326769, My BioSource, USA), CST-PathScan total Ezh2 sandwich ELISA kit (Ozyme, France), EpiQuik Dnmt1 assay kit (Euromedex/EpiGentek, France), human Bcl-2 ELISA kit (Abcam, France), caspase-2 ELISA kit (Tebu-Bio, France), and PathScan total PD-L1 sandwich ELISA kit (Ozyme, France) were performed according to the manufacturers’ instructions.

Techniques: Methylation, Expressing, Extraction, Quantitative RT-PCR, RNA Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Dot Blot, Control, Immunoprecipitation, Activity Assay, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool

doi: 10.1016/j.omtn.2020.08.010

Figure Lengend Snippet: The N6-Adenosine Methylation of miR-200b-3p Selectively Induces Apoptosis in Cancer Cells and Has an Anti-tumor Growth Effect (A) IDH mutation in U87 cells induces a decrease of the αKG rate and an increase of miR-200b-3p %m6A without FTO or METTL3 expression modification. (B) miR-200b-3p promotes cell death by itself in cancerous and non-cancerous cells (excepted neuron RN33b), while miR-200b-3b induces apoptosis by itself in U87 cells only. A lactate dehydrogenase (LDH)-cytotoxicity assay kit (Abcam, France) was used to estimate the cell death 24 h after the m6A-miR-200b-3b incubation. (C) Representation of our in vivo model of U87-induced GBM. Mice were xenografted with U87 cells. When tumor volume was equivalent to 100 mm 3 , mice were treated with TMZ and/or miR-200b-3p 5 days per week. Tumor volumes were measured after 3 weeks of treatment. (D) Impact of the adenosine-methylated form of miR-200b-3p on the tumor growth in mice model.

Article Snippet: An XIAP (human) cell-based ELISA kit (Abnova, Taiwan), αKG assay kit (no. ab83431, Abcam, France), human FTO ELISA kit (no. 68ELH-FTO, Tebu-Bio, France), METTL3 ELISA kit (no. MBS9326769, My BioSource, USA), CST-PathScan total Ezh2 sandwich ELISA kit (Ozyme, France), EpiQuik Dnmt1 assay kit (Euromedex/EpiGentek, France), human Bcl-2 ELISA kit (Abcam, France), caspase-2 ELISA kit (Tebu-Bio, France), and PathScan total PD-L1 sandwich ELISA kit (Ozyme, France) were performed according to the manufacturers’ instructions.

Techniques: Methylation, Mutagenesis, Expressing, Modification, LDH Cytotoxicity Assay, Incubation, In Vivo