Journal: Nutrients

Article Title: Roseburia intestinalis Supplementation Could Reverse the Learning and Memory Impairment and m6A Methylation Modification Decrease Caused by 27-Hydroxycholesterol in Mice

doi: 10.3390/nu16091288

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The antibodies used were as below: β-actin (ABclonal, AC026, 1:50,000, Wuhan, China), METTL4 (bioss, bs-18851R, 1:1000, Beijing, China), PSD-95 (Abcam, ab18258, 1:1000, Cambridge, UK), occludin (Abcam, ab216327, 1:1000, Cambridge, UK), claudin-1 (Abcam, ab180158, 1:1000, Cambridge, UK).

Techniques: Sequencing

Journal: Nutrients

Article Title: Roseburia intestinalis Supplementation Could Reverse the Learning and Memory Impairment and m6A Methylation Modification Decrease Caused by 27-Hydroxycholesterol in Mice

doi: 10.3390/nu16091288

Figure Lengend Snippet: Levels of the m6A modification and its related enzyme expression. ( A ) m6A methylation in the brain cortex (ng/200ng RNA). ( B ) Western blot result of METTL4. ( C ) METTL4 mRNA relative expression level in brain cortex. ( D ) METTL4 protein expression level in the brain cortex. ( E ) METTL14 mRNA relative expression level in the brain cortex, ( F ) YTHDF-1 mRNA relative expression level in the brain cortex. ( G ) FTO mRNA relative expression level in the brain cortex. ( H ) WTAP mRNA relative expression level in the brain cortex. n = 4–5 mice/group. All the data are presented as means ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. ns p > 0.05.

Article Snippet: The antibodies used were as below: β-actin (ABclonal, AC026, 1:50,000, Wuhan, China), METTL4 (bioss, bs-18851R, 1:1000, Beijing, China), PSD-95 (Abcam, ab18258, 1:1000, Cambridge, UK), occludin (Abcam, ab216327, 1:1000, Cambridge, UK), claudin-1 (Abcam, ab180158, 1:1000, Cambridge, UK).

Techniques: Modification, Expressing, Methylation, Western Blot

Journal: Translational Psychiatry

Article Title: Schizophrenia-associated differential DNA methylation in brain is distributed across the genome and annotated to MAD1L1 , a locus at which DNA methylation and transcription phenotypes share genetic variation with schizophrenia risk

doi: 10.1038/s41398-022-02071-0

Figure Lengend Snippet: Differentially methylated sites in SZ.

Article Snippet: cg04594439 , −0.025 , 0.061 , PASK.

Techniques: Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Primer Sequences used for qRT-PCR.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques:

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Inhibition of m 6 A modification confers resistance to HSC ferroptosis. METTL4 shRNA or FTO plasmid were stably transfected into HSC-LX2 cells followed by erastin (10 μM) treatment for 24 h. ( A ) The m 6 A levels were detected by m 6 A RNA Methylation Quantitative kit (**, p < 0.01, ***, p < 0.001, n = 3 in every group). ( B ) METTL4 shRNA or FTO plasmid were transfected into HSC-LX2 and HSC-T6 cells followed by sorafenib (10 μM) or erastin (10 μM) treatment for 24 h. Cell Counting Kit-8 kit was used to determine the Cell viability (*, p < 0.05, n = 3 in every group). ( C–F ) Iron accumulation, GSH depletion, lipid ROS level and MDA production were assayed (*, p < 0.05, n = 3 in every group). ( G ) FTO plasmid transfected into HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM) with or without the indicated inhibitors (Liproxstatin-1, 100 nM; Ferrostatin-1, 1 μM; Necrostatin-1, 10 μM; ZVAD-FMK, 10 μM; Necrosulfonamide, 0.5 μM) for 24 h. Cell viability was assayed by Cell Counting Kit-8. (***, p < 0.001, n = 3 in every group). ( H ) Control vector or FTO plasmid were transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h. Transmission electron microscopy was used to examin the typical changes of ferroptotic cells. Scale bars: 0.2 μm. Representative photographs were showed.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Inhibition, Modification, shRNA, Plasmid Preparation, Stable Transfection, Transfection, Methylation, Cell Counting, Control, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Reduced ferroptosis by m 6 A modification inhibition is associated with autophagy inactivation. (A) FTO plasmid was transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h. Total RNA was isolated for RNA-Seq. Clustering of HSC-LX2 cells were demonstrated by microarray heat map. The significantly differentially expressed mRNAs were analysied by hierarchical cluster: gray, no change; bright blue, underexpression; bright red, overexpression (FTO plasmid, n = 3; Control vector, n = 3). ( B ) Differentially expressed mRNAs were enriched by KEGG enrichment analysis in FTO plasmid group (Control vector, n = 3; FTO plasmid, n = 3). ( C ) The levels of m 6 A modification in autophagy-related gene were determined by MeRIP qPCR (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 3 in every group). ( D ) METTL4 shRNA or FTO plasmid transfected into HSC-T6 and HSC-LX2 cells were treated with erastin (10 μM) for 24 h. Western blot showed the protein expression of BECN1, ATG3, ATG4A, ATG7, ATG9A, ATG5-ATG12 and ATG16L1 (n = 3 in every group). ( E ) Western blot was used to determine the expression of LC3-I/II and p62 (n = 3 in every group). ( F ) METTL4 shRNA or FTO plasmid with CMV-TurboRFP- EGFP-LC3-PGK-Puro plasmid were transferred into HSC-LX2 cells by erastin (10 μM) treatment for 24 h. The fluorescence spots were detected. Representative photographs were showed. Scale bars: 50 μm. ( G ) HSC-LX2 cells transfected with FTO plasmid or control vector by erastin (10 μM) treatment for 24 h. Transmission electron microscopy was used to examine the autolysosomes or autophagosomes. Representative photographs were showed. Scale bars: 0.2 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Inhibition, Plasmid Preparation, Transfection, Isolation, RNA Sequencing, Microarray, Over Expression, Control, shRNA, Western Blot, Expressing, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Induction of autophagy by BECN1 plasmid impairs m 6 A modification inhibition-induced resistance to HSC ferroptosis. ( A ) BECN plasmid and METTL4 shRNA or FTO plasmid were transfected into HSC-T6 and HSC-LX2 cells and trested with erastin (10 μM) for 24 h. Real-time PCR were used to measure the mRNA levels of BECN1, MAPILC3B and SQSTM1 (*, p < 0.05, **, p < 0.01, compared with Control group, ## , p < 0.01, compared with BECN1 plasmid group, n = 3 in every group). ( B ) Western blot showed the protein expression of p62 and LC3-I/II (n = 3 in every group). ( C ) The endogenous LC3 levels were measured by immunofluorescence (n = 3 in every group). ( D ) HSC-LX2 and HSC-T6 cells transfected with BECN plasmid and METTL4 shRNA or FTO plasmid were treated with erastin (10 μM) or sorafenib (10 μM) for 24 h. Cell viability was assayed by Cell Counting Kit-8 (n = 3 in every group, *, p < 0.05). ( E, F ) MDA production, Iron accumulation, GSH depletion and lipid ROS level were assayed by commercial kits (*, p < 0.05, n = 3 in every group).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Plasmid Preparation, Modification, Inhibition, shRNA, Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing, Immunofluorescence, Cell Counting

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: HSC-specific inhibition of m 6 A modification impairs erastin-induced HSC ferroptosis in murine liver fibrosis. Mice of 6 groups were treated with Vehicle, CCl 4, CCl 4 +VA-Lip-control-vector + Erastin, CCl 4 +VA-Lip-Mettl4-shRNA + Erastin, CCl 4 +VA-Lip-Fto-Plasmid + Erastin, CCl 4 +VA-Lip-Ythdf1-shRNA + Erastin. ( A ) Macroscopic examination was used to observe the pathological changes of the livers. Scale bars: 1 cm. Histopathological study was performed by H&E, Masson, and Sirius Red staining. Representative photographs were showed. Scale bars: 50 μm. (***, p < 0.001, n = 6 in every group). ( B ) Immunohistochemical staining of α-SMA was determined. Representative photographs were showed. Scale bars: 50 μm.(***, p < 0.001, n = 6 in every group). ( C ) Real-time PCR was measured to determine the mRNA expression of liver fibrosis markers (Acta2, Col1a1, Fn1, and Des) (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 6 in every group). ( D ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (n = 6 in every group, ***, p < 0.001, N.S., not significant). ( E, F ) Real-time PCR was used to determine the mRNA expression of autophagy markers (Becn1, Map1lc3b, Sqstm1, and Fth1) (*, p < 0.05, ***, p < 0.001, N.S., not significant, n = 6 in every group). ( G ) The mRNA expression of Ptgs2, iron accumulation and MDA production were determined (*, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant, n = 6 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Inhibition, Modification, Control, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Expressing, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification upregulation, autophagy activation, and ferroptosis induction occur in human HSCs receiving sorafenib monotherapy. ( A, B ) laser capture microdissection (LCM) was used to isolated the primary human HSCs from the collected liver tissue. ACTA2, FN1, COL1A1, METTL4, FTO, and YTHDF1 mRNA expression were determined by real-time PCR (No treatment, n = 10; Sorafenib treatment, n = 10, **, p < 0.01, ***, p < 0.001). ( C ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( D ) Real-time PCR was used to determine the mRNA expression of autophagy markers BECN1, MAP1LC3B, SQSTM1, and FTH1 (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( E ) The PTGS2 mRNA expression, iron accumulation, MDA production and GSH depletion were determined (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Activation Assay, Laser Capture Microdissection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification induces HSC ferroptosis by regulating autophagy signaling pathway. The upregulation of methylase METTL4 and the downregulation of demethylase FTO increased the levels of m 6 A modifications in BECN1 mRNA. m 6 A reader YTHDF1 promoted BECN1 mRNA stability via recognizing the m 6 A binding site, thus triggering autophagy activation, and eventually leading to HSC ferroptosis.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Binding Assay, Activation Assay

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: p3xFlag-Mettl4 , This study , Addgene plasmid 86665.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Mettl4 and Alkbh4 Catalyze Deposition and Erasure, Respectively, of 6mA (A) Schematic presentation of Mettl4 protein structure and linear arrangement of conserved motifs in methyltransferase domain (amino acids 257–471), showing detailed alignment to motif IV sequence logo in catalytic site of MT-A70 adenine N6-methyltransferases. (B) Clustering of proteins within the MT-A70 family. RNA-specific methyltransferases form a tight cluster of closely related sequences, while Mettl4 and the DNA adenine N6-methyltransferases DAMT-1 ( C. elegans ) and M. MunI (Mycoplasma) (red circles) are positioned apart. (C) HEK293T cells transfected with empty vector (EV) or expressing Flag-Dam or the wild-type or DPPW catalytic-site mutant (PPmut) variants of Flag-Mettl4 were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with Flag antisera. Actin, loading control. 6mA and 5mC in the same samples were detected by dot blot of genomic DNA (n = 3 experiments), and mean 6mA levels relative to dA were quantified by LC-MS/MS analysis of genomic DNA hydrolysates (n = 2 experiments). (D) Levels of 6mA relative to total dA in genomic DNA (left panel) and m6A relative to total adenosine (A) in mRNA (right panel), purified from WT and Mettl4 KO ESCs, as determined by quantitative LC-MS/MS. Dashed line represents limit of detection. n.d., not detected. Data are plotted as mean with SD (n = 2 experiments). (E) Overlaid extracted LC-MS chromatograms of dA and 6mA in genomic DNA hydrolysates prepared from WT and Mettl4 KO spleens. (F) In vitro 6mA demethylation assay. Recombinant His-Alkbh4 was purified from bacterial cell extracts (left panel). Irrelevant lanes were omitted from the gel. Human genomic DNA containing 6mA was incubated in buffer supplemented with Fe 2+ and 2-oxoglutarate cofactors in the absence or presence of His-Alkbh4 or vitamin C (VitC) for the indicated times. 6mA in each sample was detected by dot blot analysis (n = 3 experiments). (G) Alkbh4 demethylates 6mA in double-stranded (ds) DNA. Single-stranded (ss) or double-stranded DNA oligonucleotide containing unmodified adenine or 6mA was incubated in the absence or presence of His-Alkbh4 for the indicated times. Following the reactions, 6mA in 10 pmol of each DNA sample was detected by dot blot analysis (n = 3 experiments). See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Western Blot, Control, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Purification, In Vitro, Demethylation Assay, Recombinant, Incubation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Embryonic Sublethality and Craniofacial Dysmorphism in Mettl4 KO Incross Progeny (A) Genotypic analysis of progeny from intercrossed Mettl4 ± heterozygous (Het) mice, showing expected and observed frequencies for each genotype. n.s., not significant, Chi-square test. (B) Mating scores of strain-matched wild-type (WT) controls, intercrossed Mettl4 ± heterozygotes, and incrossed Mettl4 KO mice. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by Caesarian section at E18.5. Red lines indicate the mean litter size. p value was calculated by two-tailed t test. ∗∗ p < 0.01. The percentages of progeny that displayed craniofacial or limb dysmorphism are shown below each plot. (C) Neonatal Mettl4 KO pups from a single litter of incrossed Mettl4 KO parents. White arrow indicates mandibular malformation. Scale bar, 10 mm. (D) Representative image of spleens recovered from individual adult (10–12 weeks old) WT or Mettl4 KO mice. Rulers show scale in cm. Separate images for WT and KO spleens were juxtaposed at the dashed line. (E) Bar graph representation of spleen mass divided by total body mass for adult WT and Mettl4 KO mice (n = 6 mice per group). Error bars indicate SD ∗∗ p < 0.01, two-tailed t test. (F) (Left panel) Peripheral blood count of white cells (WBC) and lymphocytes (left-hand scale) and red blood cells (RBC, right-hand scale) in adult WT and Mettl4 KO mice. (Right panel) Hematocrit (HCT) analysis. Each point corresponds to values from an individual mouse. Brackets indicate normal range and mean values in WT mice. p values were calculated using unpaired, two-tailed t test. ∗ p < 0.05. See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Two Tailed Test

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND (A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain. (B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments). (C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments). (D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments). (E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments). (F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments). See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: In Vitro, Purification, Recombinant, Incubation, SDS Page, Western Blot, Expressing, Plasmid Preparation, Dot Blot, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: The E3 Ubiquitin Ligase TRIP12 Mediates Proteolysis of ASXL1 (A) Lysates prepared from HEK293T cells expressing the indicated combinations of Flag-ASXL1.591, Flag-Mettl4, or GFP-TRIP12 were immunoprecipitated using TRIP12 antisera or isotype-matched control IgG. Inputs and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotting with Flag or TRIP12 antisera (n = 2 experiments). (B) HEK293T cells expressing Flag-ASXL1.591 and either a control shRNA targeting GFP or independent shRNAs (sh1 or sh2) targeting TRIP12 were lysed and protein extracts resolved by SDS-PAGE and immunoblotting. Tubulin, loading control (n = 3 experiments). (C) Protein extracts prepared from cells expressing Flag-ASXL1.591, in the absence or presence of Flag-Mettl4 or sh1- TRIP12 , were resolved by SDS-PAGE and immunoblotting with the indicated antisera (n = 3 experiments). (D) Model depicting 6mA deposition by Mettl4, recruitment of ASXL1/PR-DUB to 6mA, and engagement of PR-DUB with the E3 ubiquitin ligase TRIP12. These steps are proposed to stimulate ASXL1 proteolysis, inactivating PR-DUB and thereby preserving the Polycomb repressive mark H2A-K119Ub in chromatin.

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, shRNA, Preserving

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Ectopic Asxl1 and Mpnd Correspond with Loss of Polycomb Silencing in Mettl4 KO ESCs (A) Pie chart illustrating the distribution of called 6mA peaks (n = 4,922) across promoter (−2 kb to TSS), TSS downstream (0–2 kb downstream of TSS), 5′ and 3′ UTR, coding exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of 6mA at each feature relative to a random distribution. (B) Venn diagram showing overlap of 6mA peaks identified by MeDIP and called 6mA bases identified by PacBio SMRT sequencing analysis ( Wu et al., 2016 ). p value, Fisher’s exact test. (C) Normalized 6mA tag density plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (blue), and the bottom 10% of least expressed genes in WT ESCs (red). (D) Venn diagrams showing the number and relative distribution of Asxl1, Bap1, O-GlcNAc, Mpnd, and H2A-K119Ub (H2A-Ub) peaks in WT and Mettl4 KO ESCs. (E) Normalized tag density of ectopic Asxl1 peaks induced in Mettl4 KO ESCs (left) with heatmap representation of peaks ranked-ordered by the mean signal (right), each plotted across a window centered on the TSS. (F) Genome browser view showing profiles of 6mA or isotype-matched control IgG in WT ESCs (top) and Asxl1, Mpnd, H2A-Ub, Bap1, and O-GlcNAc in WT and Mettl4 KO ESCs at two representative loci. Shaded vertical bars highlight regions containing ectopic Asxl1 or Mpnd and depletion of H2A-Ub in Mettl4 KO ESCs. RefSeq exon structures (blue) for each annotated gene are shown at the bottom. (G) ChIP-qPCR analysis of the Rpl13 and Dvl3 genes in WT and Mettl4 KO ESCs. Antisera specific for Asxl1, H2A-K119Ub002C or Bap1 were used for chromatin immunoprecipitation. The mean fold enrichments normalized to isotype-matched IgG control are shown for each condition. Error bars indicate SEM (n = 2 experiments). (H) RT-qPCR analysis of Rpl13 and Dvl3 transcript levels in WT and Mettl4 KO ESCs. The mean value of WT control samples is set as 1. Error bars indicate SEM (n = 2 experiments). (I) Volcano plot presentation of transcript levels for genes expressed in WT and Mettl4 KO ESCs as determined by RNA-seq. Genes strongly up- or downregulated (FC > 2.0, FDR-adjusted p < 0.05) in Mettl4 KO cells are identified and indicated in red or green, respectively. (J) Gene ontology analysis of differentially expressed genes induced in Mettl4 KO cells showing involvement in embryonic development and tissue patterning. The yellow vertical line indicates the threshold for significance. See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Methylated DNA Immunoprecipitation, Sequencing, Control, ChIP-qPCR, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Sequencing

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Mettl4 and Alkbh4 Catalyze Deposition and Erasure, Respectively, of 6mA (A) Schematic presentation of Mettl4 protein structure and linear arrangement of conserved motifs in methyltransferase domain (amino acids 257–471), showing detailed alignment to motif IV sequence logo in catalytic site of MT-A70 adenine N6-methyltransferases. (B) Clustering of proteins within the MT-A70 family. RNA-specific methyltransferases form a tight cluster of closely related sequences, while Mettl4 and the DNA adenine N6-methyltransferases DAMT-1 ( C. elegans ) and M. MunI (Mycoplasma) (red circles) are positioned apart. (C) HEK293T cells transfected with empty vector (EV) or expressing Flag-Dam or the wild-type or DPPW catalytic-site mutant (PPmut) variants of Flag-Mettl4 were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with Flag antisera. Actin, loading control. 6mA and 5mC in the same samples were detected by dot blot of genomic DNA (n = 3 experiments), and mean 6mA levels relative to dA were quantified by LC-MS/MS analysis of genomic DNA hydrolysates (n = 2 experiments). (D) Levels of 6mA relative to total dA in genomic DNA (left panel) and m6A relative to total adenosine (A) in mRNA (right panel), purified from WT and Mettl4 KO ESCs, as determined by quantitative LC-MS/MS. Dashed line represents limit of detection. n.d., not detected. Data are plotted as mean with SD (n = 2 experiments). (E) Overlaid extracted LC-MS chromatograms of dA and 6mA in genomic DNA hydrolysates prepared from WT and Mettl4 KO spleens. (F) In vitro 6mA demethylation assay. Recombinant His-Alkbh4 was purified from bacterial cell extracts (left panel). Irrelevant lanes were omitted from the gel. Human genomic DNA containing 6mA was incubated in buffer supplemented with Fe 2+ and 2-oxoglutarate cofactors in the absence or presence of His-Alkbh4 or vitamin C (VitC) for the indicated times. 6mA in each sample was detected by dot blot analysis (n = 3 experiments). (G) Alkbh4 demethylates 6mA in double-stranded (ds) DNA. Single-stranded (ss) or double-stranded DNA oligonucleotide containing unmodified adenine or 6mA was incubated in the absence or presence of His-Alkbh4 for the indicated times. Following the reactions, 6mA in 10 pmol of each DNA sample was detected by dot blot analysis (n = 3 experiments). See also and .

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Western Blot, Control, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Purification, In Vitro, Demethylation Assay, Recombinant, Incubation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Embryonic Sublethality and Craniofacial Dysmorphism in Mettl4 KO Incross Progeny (A) Genotypic analysis of progeny from intercrossed Mettl4 ± heterozygous (Het) mice, showing expected and observed frequencies for each genotype. n.s., not significant, Chi-square test. (B) Mating scores of strain-matched wild-type (WT) controls, intercrossed Mettl4 ± heterozygotes, and incrossed Mettl4 KO mice. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by Caesarian section at E18.5. Red lines indicate the mean litter size. p value was calculated by two-tailed t test. ∗∗ p < 0.01. The percentages of progeny that displayed craniofacial or limb dysmorphism are shown below each plot. (C) Neonatal Mettl4 KO pups from a single litter of incrossed Mettl4 KO parents. White arrow indicates mandibular malformation. Scale bar, 10 mm. (D) Representative image of spleens recovered from individual adult (10–12 weeks old) WT or Mettl4 KO mice. Rulers show scale in cm. Separate images for WT and KO spleens were juxtaposed at the dashed line. (E) Bar graph representation of spleen mass divided by total body mass for adult WT and Mettl4 KO mice (n = 6 mice per group). Error bars indicate SD ∗∗ p < 0.01, two-tailed t test. (F) (Left panel) Peripheral blood count of white cells (WBC) and lymphocytes (left-hand scale) and red blood cells (RBC, right-hand scale) in adult WT and Mettl4 KO mice. (Right panel) Hematocrit (HCT) analysis. Each point corresponds to values from an individual mouse. Brackets indicate normal range and mean values in WT mice. p values were calculated using unpaired, two-tailed t test. ∗ p < 0.05. See also and .

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: Two Tailed Test

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND (A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain. (B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments). (C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments). (D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments). (E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments). (F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments). See also and .

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: In Vitro, Purification, Recombinant, Incubation, SDS Page, Western Blot, Expressing, Plasmid Preparation, Dot Blot, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: The E3 Ubiquitin Ligase TRIP12 Mediates Proteolysis of ASXL1 (A) Lysates prepared from HEK293T cells expressing the indicated combinations of Flag-ASXL1.591, Flag-Mettl4, or GFP-TRIP12 were immunoprecipitated using TRIP12 antisera or isotype-matched control IgG. Inputs and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotting with Flag or TRIP12 antisera (n = 2 experiments). (B) HEK293T cells expressing Flag-ASXL1.591 and either a control shRNA targeting GFP or independent shRNAs (sh1 or sh2) targeting TRIP12 were lysed and protein extracts resolved by SDS-PAGE and immunoblotting. Tubulin, loading control (n = 3 experiments). (C) Protein extracts prepared from cells expressing Flag-ASXL1.591, in the absence or presence of Flag-Mettl4 or sh1- TRIP12 , were resolved by SDS-PAGE and immunoblotting with the indicated antisera (n = 3 experiments). (D) Model depicting 6mA deposition by Mettl4, recruitment of ASXL1/PR-DUB to 6mA, and engagement of PR-DUB with the E3 ubiquitin ligase TRIP12. These steps are proposed to stimulate ASXL1 proteolysis, inactivating PR-DUB and thereby preserving the Polycomb repressive mark H2A-K119Ub in chromatin.

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, shRNA, Preserving

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Ectopic Asxl1 and Mpnd Correspond with Loss of Polycomb Silencing in Mettl4 KO ESCs (A) Pie chart illustrating the distribution of called 6mA peaks (n = 4,922) across promoter (−2 kb to TSS), TSS downstream (0–2 kb downstream of TSS), 5′ and 3′ UTR, coding exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of 6mA at each feature relative to a random distribution. (B) Venn diagram showing overlap of 6mA peaks identified by MeDIP and called 6mA bases identified by PacBio SMRT sequencing analysis ( Wu et al., 2016 ). p value, Fisher’s exact test. (C) Normalized 6mA tag density plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (blue), and the bottom 10% of least expressed genes in WT ESCs (red). (D) Venn diagrams showing the number and relative distribution of Asxl1, Bap1, O-GlcNAc, Mpnd, and H2A-K119Ub (H2A-Ub) peaks in WT and Mettl4 KO ESCs. (E) Normalized tag density of ectopic Asxl1 peaks induced in Mettl4 KO ESCs (left) with heatmap representation of peaks ranked-ordered by the mean signal (right), each plotted across a window centered on the TSS. (F) Genome browser view showing profiles of 6mA or isotype-matched control IgG in WT ESCs (top) and Asxl1, Mpnd, H2A-Ub, Bap1, and O-GlcNAc in WT and Mettl4 KO ESCs at two representative loci. Shaded vertical bars highlight regions containing ectopic Asxl1 or Mpnd and depletion of H2A-Ub in Mettl4 KO ESCs. RefSeq exon structures (blue) for each annotated gene are shown at the bottom. (G) ChIP-qPCR analysis of the Rpl13 and Dvl3 genes in WT and Mettl4 KO ESCs. Antisera specific for Asxl1, H2A-K119Ub002C or Bap1 were used for chromatin immunoprecipitation. The mean fold enrichments normalized to isotype-matched IgG control are shown for each condition. Error bars indicate SEM (n = 2 experiments). (H) RT-qPCR analysis of Rpl13 and Dvl3 transcript levels in WT and Mettl4 KO ESCs. The mean value of WT control samples is set as 1. Error bars indicate SEM (n = 2 experiments). (I) Volcano plot presentation of transcript levels for genes expressed in WT and Mettl4 KO ESCs as determined by RNA-seq. Genes strongly up- or downregulated (FC > 2.0, FDR-adjusted p < 0.05) in Mettl4 KO cells are identified and indicated in red or green, respectively. (J) Gene ontology analysis of differentially expressed genes induced in Mettl4 KO cells showing involvement in embryonic development and tissue patterning. The yellow vertical line indicates the threshold for significance. See also and .

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: Methylated DNA Immunoprecipitation, Sequencing, Control, ChIP-qPCR, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Sequencing

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: Mettl4 CRISPR/Cas9 KO , Santa Cruz Biotechnology , Cat#sc-429415.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Scientific Reports

Article Title: METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

doi: 10.1038/s41598-023-44379-x

Figure Lengend Snippet: Antibodies used in the study are shown.

Article Snippet: METTL14 , 1: 1000 , Rabbit , 55 , orb28269, Biorbyt.

Techniques:

Journal: Scientific Reports

Article Title: METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

doi: 10.1038/s41598-023-44379-x

Figure Lengend Snippet: METTL3 mediated m6A methylation of STAG3. ( A ) In CRC and adjacent normal tissues, the protein expression levels of METTL3, METTL14, ALKBH5, and FTO were determined by WB. ( B,C ) The mRNA and protein expression levels of METTL3 in HCT116 cells were measured using qRT-PCR and WB. ( D ) The STAG3 m6A modification level in HCT116 cells was evaluated by Me-RIP. *P < 0.05 vs. normal, & P < 0.05 vs. sh-NC, # P < 0.05 vs. oe-NC. Superscript a: P < 0.05, superscript b: P < 0.01, superscript d: P < 0.0001.

Article Snippet: METTL14 , 1: 1000 , Rabbit , 55 , orb28269, Biorbyt.

Techniques: Methylation, Expressing, Quantitative RT-PCR, Modification

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: METTL4 (methyltransferase-like protein 4) is involved in the progression of atherosclerosis. A and B , N6-methyldeoxyadenosine (6mA) dot blot ( A ) and MethylFlash m6A DNA Methylation ELISA Kit ( B ) analysis of mitochondrial DNA (mtDNA) 6mA levels in human monocyte–derived macrophages (HMDMs) treated with or without oxidized low-density lipoprotein (ox-LDL, 50 μg/mL, 24 hours). n=6 per group. C , 6mA dot blot analysis of mtDNA and total DNA in ox-LDL–stimulated HMDMs. n=6 per group. D , Western blot analysis of the subcellular localization of METTL4 protein in HMDMs treated with or without ox-LDL. VDAC (mitochondria) and H3 (nucleus) were selected as organelle-specific marker proteins. n=6 per group. E , Super-resolution fluorescence imaging the colocalization of METTL4 (red) with mitochondria (TOMM20, green) in ox-LDL–stimulated HMDMs with Scr or si METTL4 . n=6 per group. F , 6mA dot blot analysis of mtDNA 6mA levels in HMDMs transfected with Scr or si METTL4 followed by ox-LDL stimulation. n=6 per group. G , Western blot analysis of the levels of METTL4 in nonatherosclerotic (Non-AS) and atherosclerotic (AS) arteries derived from patients. n=6 per group. H , Western blot analysis of the levels of METTL4 in the arteries derived from Apoe -/ - mice fed with normal chow (NC) or high-fat diet (HFD). n=6 per group. I , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of Mettl4 and inflammatory factors ( MCP-1 , IL-1β , and TNF-α ) in the athero-prone (lower curvature, LC) and athero-protective (greater curvature, GC) regions of atherosclerotic lesions derived from HFD-fed Apoe -/ - mice. n=6 per group. J , Western blot analysis of METTL4 in human aortic smooth muscle cells (HASMCs), human aortic endothelial cells (HAECs), HMDMs, and bone marrow–derived macrophages (BMDMs) treated with or without ox-LDL. n=6 per group. K , Immunofluorescence analysis of METTL4 (green) and macrophage marker (CD68, red) in the HMDMs treated with or without ox-LDL. n=6 per group. L , Immunofluorescence analysis of METTL4 (green) and macrophage marker (CD68, red) in the aortic root from Apoe -/ - mice fed with a HFD for 8 and 12 weeks. n=6 per group. M , RT-qPCR analysis of METTL4 levels in peripheral blood mononuclear cells (PBMCs) of healthy individuals and PBMCs of symptomatic and asymptomatic patients with carotid atherosclerosis. n=21 per group. N , Linear regression analysis of mRNA levels of METTL4 with inflammatory factors in PBMCs from patients with carotid atherosclerosis. n=42 per group. O , Nuclear run-on experiments coupled with RT-qPCR analysis of the global nascent METTL4 transcript in HMDMs treated with or without ox-LDL. n=6 per group. Data represent the mean±SEM. ** P <0.01, *** P <0.001 by unpaired 2-sided Student t test ( B and H through J ), unpaired 2-sided Student t test with Welch correction ( I and O ), Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multiple comparisons test ( M ), Mann-Whitney test ( G ), and Pearson correlation analysis ( N ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Dot Blot, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Marker, Fluorescence, Imaging, Transfection, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, MANN-WHITNEY

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: Myeloid-specific deletion of METTL4 reduces atherosclerosis. A , Strategy for the generation of Mettl4 flox/flox mice. B , Schematic diagram of atherosclerotic model establishment in Mettl4 flox/flox -Apoe -/ - mice and Mettl4 Mac-KO -Apoe -/ - mice. C , En face Oil Red O staining of the aortas of HFD-fed Mettl4 flox/flox -Apoe -/ - mice and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. D and E , The Oil Red O ( D ) and HE staining ( E ) in the aortic roots of HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. F , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of CD11b within the aortic root plaques derived from HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=10 per group. G , Representative immunofluorescence staining images of macrophages (CD68, red) and DAPI (blue) in the aortic roots from HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. H , The Masson and Sirius red staining in the aortic roots of HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. I , Representative immunofluorescence staining images of smooth muscle cell (SMA, green) and DAPI (blue) in the aortic roots of HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. J , Dot blot analysis of mtDNA 6mA levels in thioglycolate-elicited peritoneal macrophages (TEPMs) derived from HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. Data represent the mean±SEM. ** P <0.01, *** P <0.001 by unpaired 2-sided Student t test ( C through E and H ) and unpaired 2-sided Student t test with Welch correction ( C and F through I ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Staining, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Derivative Assay, Immunofluorescence, Dot Blot

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: METTL4-activated macrophage inflammasome through cytoplasmic mtDNA released through mitochondrial permeability transition pore (mPTP) opening. A , Volcano plot of RNA-Seq data (GSE280434) from HMDMs treated with si METTL4 +ox-LDL and Scr+ox-LDL. B , Bubble diagram illustrating the Gene Ontology (GO) enrichment of differentially expressed genes. Each circle corresponds to the number of genes assigned to each category. C , Enrichment of genes involved in GO biological processes. D , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of inflammatory factors in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation is presented as a heatmap. n=6 per group. E , Flow cytometry analysis of mitochondrial membrane potential using a JC-1 probe in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. F , Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation were monitored using a Seahorse XFe24 analyzer. n=6 per group. G , The ultrastructure of mitochondria in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation was examined by transmission electron microscopy. LD indicates lipid droplets; M, mitochondria; and N, nucleus. n=6 per group. H , The fluorescence imaging of mitochondrial morphology in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. I , Quantitative PCR (qPCR) analysis of cytoplasmic mtDNA ( MT-ND1 and MT-ND2 ), nuclear LINE1 elements ( L1ORF1 and L1ORF2 ), and ribosomal gene ( 18SRRNA ) in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. J , The METTL4-deficient HMDMs were transfected with or without METTL4 overexpression plasmid, which were then pretreated with 5 μM CsA for 2 hours, followed by ox-LDL stimulation. Then, qPCR was conducted to analyze cytoplasmic DNA content of these HMDMs. n=6 per group. K , Western blot analysis of inflammasome-associated proteins (caspase-1 and IL-1β) in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. L , The METTL4-deficient HMDMs were transfected with or without METTL4 overexpression plasmid, which were then treated with mtDNA or DNase (deoxyribonuclease) I. Then, Western blot was conducted to analyze the expression of inflammasome-associated proteins in the cells. n=6 per group. M , OCR and ECAR in TEPMs from HFD-fed Mettl4 Mac-KO -Apoe -/ - and Mettl4 flox/flox -Apoe -/ - mice were monitored using a Seahorse XFe24 analyzer. n=6 per group. N , Representative immunofluorescence staining images of macrophage (CD68, red) and IL-1β (green) in the aortic roots derived from HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. n=6 per group. O , RT-qPCR analysis of inflammatory factors in plaque macrophages from HFD-fed Mettl4 flox/flox -Apoe -/ - and Mettl4 Mac-KO -Apoe -/ - mice. The results are presented as a heatmap. n=6 per group. Data represent the mean±SEM. * P <0.05, ** P <0.01, *** P <0.001 by 2-way ANOVA followed by Tukey multiple comparisons test ( D and I ), Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multiple comparisons test ( J ), unpaired 2-sided Student t test ( O ), and unpaired 2-sided Student t test with Welch correction ( O ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Permeability, RNA Sequencing, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Transfection, Flow Cytometry, Membrane, Transmission Assay, Electron Microscopy, Fluorescence, Imaging, Real-time Polymerase Chain Reaction, Over Expression, Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Staining, Derivative Assay

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: The mtDNA 6mA is the critical factor for METTL4 in regulating mitochondrial dysfunction in ox-LDL–stimulated macrophages. A , MethylFlash m6A DNA Methylation ELISA Kit analysis of mtDNA 6mA levels in HMDMs transfected with pcDNA, METTL4-WT, and METTL4-MUT followed by ox-LDL stimulation. n=6 per group. B , qPCR (quantitative polymerase chain reaction) analysis of cytoplasmic mtDNA content in HMDMs transfected with pcDNA, METTL4-WT, and METTL4-MUT followed by ox-LDL stimulation. n=6 per group. C , Flow cytometry analysis of the mitochondrial membrane potential using the JC-1 probe in METTL4-deficient HMDMs transfected with METTL4-WT and METTL4-MUT followed by ox-LDL stimulation. n=6 per group. D , ECAR and OCR of the METTL4-deficient HMDMs transfected with METTL4-WT and METTL4-MUT followed by ox-LDL stimulation were monitored using a Seahorse XFe24 analyzer. n=6 per group. We constructed mtDNA-depleted METTL4-deficient HMDMs (ρ0) using Etbr. Then, the ρ0 cells and METTL4-deficient HMDMs were transfected with METTL4-WT or METTL4-MUT, followed by ox-LDL stimulation. E , Transmission electron microscopy analysis of the ultrastructure of HMDMs. LD indicates lipid droplets; M, mitochondria; and N, nucleus. n=6 per group. F , Western blot analysis of inflammasome-associated proteins in HMDMs. n=6 per group. G , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of inflammatory factors in HMDMs. The results are presented as a heatmap. n=6 per group. H , Flow chart of experimental procedure for mitochondrial transplantation in HMDMs. I , Western blot analysis of the purity of extracted mitochondria. n=6 per group. J , Flow cytometry analysis of the internalization of MitoTracker Green–labeled mitochondria in HMDMs. n=3 per group. The mitochondria were extracted from HMDMs transfected with METTL4-WT followed by ox-LDL stimulation (Mit WT+ox-LDL ) or HMDMs transfected with METTL4-MUT followed by ox-LDL stimulation (Mit MUT+ox-LDL ). Then, the METTL4-deficient HMDMs were transfected with pcDNA followed by ox-LDL stimulation, which were then transplanted with Mit WT+ox-LDL . The METTL4-deficient HMDMs were transfected with METTL4 overexpression plasmid followed by ox-LDL stimulation, which were then transplanted with Mit MUT+ox-LDL . K , The effects of mitochondrial transplantation on ECAR and OCR in HMDMs were monitored using a Seahorse XFe24 analyzer. n=6 per group. L , TMRM fluorescence staining analysis showing the effects of mitochondrial transplantation on mitochondrial membrane potential. n=6 per group. M , MethylFlash m6A DNA Methylation ELISA Kit analysis of the effect of mitochondrial transplantation on mtDNA 6mA levels in HMDMs. n=6 per group. N , qPCR analysis of the effects of mitochondrial transplantation on the cytoplasmic mtDNA content in HMDMs. n=6 per group. O , RT-qPCR analysis of the effects of mitochondrial transplantation on expressions of inflammatory factors in HMDMs. n=6 per group. Data represent the mean±SEM. * P <0.05, ** P <0.01, *** P <0.001 by 1-way ANOVA followed by Tukey multiple comparisons test ( A , G , M , and O ), and Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multiple comparisons test ( B , L , N , and O ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Flow Cytometry, Membrane, Construct, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Transplantation Assay, Labeling, Over Expression, Plasmid Preparation, Fluorescence, Staining

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: METTL4-mediated MT-ATP6 6mA caused excess protons accumulated in the mitochondrial intermembrane space. A , Western blot analysis of mitochondrial respiratory chain complex I-V in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. B , Mitochondrial respiration chain complex activity assay kit measures the activity of mitochondrial respiratory chain complex I-V in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. C , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of mtDNA encoded genes in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation, as shown in the heat map. n=6 per group. D , Western blot analysis of MT-ATP6 expression in HMDMs transfected with or without si METTL4 followed by ox-LDL stimulation. n=6 per group. E , RT-qPCR analysis of MT-ATP6 in METTL4-deficient HMDMs transfected with pcDNA, METTL4-WT, or METTL4-MUT followed by ox-LDL stimulation. n=6 per group. F , Western blot analysis of MT-ATP6 in METTL4-deficient HMDMs transfected with pcDNA, METTL4-WT, or METTL4-MUT followed by ox-LDL stimulation. n=6 per group. G , The mtDNA 6mA motif of MT-ATP6 ( left ). Immunoprecipitation (IP) analysis of the binding of 6mA and MT-ATP6 in METTL4-deficient HMDMs transfected with pcDNA, METTL4-WT, or METTL4-MUT followed by ox-LDL stimulation ( right ). n=6 per group. H , IP analysis of the binding of TFAM and MT-ATP6 in METTL4-deficient HMDMs transfected with pcDNA, METTL4-WT, or METTL4-MUT followed by ox-LDL stimulation. n=6 per group. I through L , The analysis of the ECAR and OCR ( I ), cytoplasmic mtDNA content ( J ), the expression of inflammasome-associated proteins ( K ), and the inflammation ( L ) in METTL4-deficient HMDMs transfected with METTL4 or ATP6 overexpression plasmid followed by ox-LDL stimulation. n=6 per group. M , Fluorescence analysis of the submitochondrial localization of protons in METTL4-deficient HMDMs transfected with METTL4 or ATP6 overexpression plasmid followed by ox-LDL stimulation. Mitochondrial (TOMM20, green) and protons (red) are shown. n=6 per group. Data represent the mean±SEM. ** P <0.01, *** P <0.001 by unpaired 2-sided Student t test ( A through C ), unpaired 2-sided Student t test with Welch correction ( B ), 1-way ANOVA followed by Tukey multiple comparisons test ( E and H ), Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multiple comparisons test ( G , J , and L ), and Kruskal-Wallis test followed by Dunn multiple comparisons test ( M ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Western Blot, Transfection, Activity Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Expressing, Immunoprecipitation, Binding Assay, Over Expression, Plasmid Preparation, Fluorescence

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: Myeloid-specific mutation in METTL4 methyltransferase active site reduced atherosclerosis. A , En face Oil Red O staining of the aortas of HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. B , MethylFlash m6A DNA Methylation ELISA Kit analysis of mtDNA 6mA levels in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=10 per group. C , Oil Red O and HE staining of aortic roots of HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. D , Representative immunofluorescence staining images of macrophage (CD68, red) and DAPI staining (blue) in the aortic roots of HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. E , Masson, Sirius red, and immunofluorescence staining images of smooth muscle cells (SMA, green) and DAPI (blue) in the aortic roots of HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. F , RT-qPCR (quantitative polymerase chain reaction) analysis of inflammatory factors in plaque macrophages from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. G , IP analysis of 6mA and MT-ATP6 binding in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=10 per group. H and I , RT-qPCR (quantitative reverse transcription polymerase chain reaction) and Western blot analysis of the expression of MT-ATP6 in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6–10 per group. J , Mitochondrial respiration chain complex activity assay kit analysis of the activity of mitochondrial respiratory chain complex I-V in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. K , ECAR and OCR in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice were monitored using a Seahorse XFe24 analyzer. n=6 per group. L , qPCR analysis of cytoplasmic mtDNA content in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - mice and Mettl4 MUT -Apoe -/ - mice. n=10 per group. M , Western blot analysis of inflammasome-associated proteins in TEPMs from HFD-fed Mettl4 WT -Apoe -/ - and Mettl4 MUT -Apoe -/ - mice. n=6 per group. N , En face Oil Red O staining of the aorta of Mettl4 Mac-KO -Apoe -/ - mice received bone marrow from Mettl4 Mac-KO -Apoe -/ - (KO), Mettl4 WT -Apoe -/ - (WT), or Mettl4 MUT -Apoe -/ - (MUT) mice followed by a HFD. n=6 per group. O , RT-qPCR analysis of CD11b within the aortic root plaques from HFD-fed Mettl4 Mac-KO -Apoe -/ - mice receiving bone marrow cell transplantation treated as in N . n=10 per group. P , RT-qPCR analysis of inflammatory factors in plaque macrophages from Mettl4 Mac-KO -Apoe -/ - mice receiving bone marrow cell transplantation treated as in N . n=10 per group. Data represent the mean±SEM. ** P <0.01, *** P <0.001 by unpaired 2-sided Student t test ( A through D , F , H , and J ), unpaired 2-sided Student t test with Welch correction ( A , F , G , I , and L ), Brown-Forsythe and Welch ANOVA test followed by Dunnett T3 multiple comparisons test ( N ), Kruskal-Wallis test followed by Dunn multiple comparisons test ( O and P ), and 1-way ANOVA followed by Tukey multiple comparisons test ( P ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Mutagenesis, Staining, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Expressing, Activity Assay, Transplantation Assay

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: Pemetrexed (PEM) was identified as the first METTL4 antagonist effective in mitigating atherosclerosis progression. A , Procedure for 3D mathing and ensemble docking-based virtual screening to identify METTL4 inhibitors. B , Enzymatic inhibitory activity of the indicated compounds against purified METTL4 using a bioluminescence assay. n=6 per group. C , The molecular docking of Pemetrexed, Xanthinol Nicotinate, and Linagliptin with METTL4. D , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of MT-ATP6 expression in ox-LDL–stimulated HMDMs treated with PEM, Xanthinol Nicotinate (XN), and Linagliptin. n=6 per group. E , RT-qPCR analysis of inflammatory factors in ox-LDL–stimulated HMDMs treated with PEM, XN, and Linagliptin. The results are presented as a heatmap. n=6 per group. F , RT-qPCR analysis of MT-ATP6 in ox-LDL–stimulated HMDMs treated with different concentrations of PEM. n=6 per group. G , IP analysis of 6mA and MT-ATP6 binding in ox-LDL–stimulated HMDMs treated with or without PEM. n=6 per group. H , En face Oil Red O staining of the aortas of HFD-fed Apoe -/ - mice treated with different dosages of PEM. n=6 per group. I , Oil Red O staining of the aortic roots of HFD-fed Apoe -/ - mice treated with different dosages of PEM. n=6 per group. J , The HE, Masson, and Sirius red staining of the aortic roots of HFD-fed Apoe -/ - mice with or without PEM. n=6 per group. K and L , Dot blot analysis of mtDNA 6mA levels ( K ) and Western blot analysis of MT-ATP6 expression ( L ) in TEPMs from HFD-fed Apoe -/ - mice with or without PEM. n=6 per group. M , qPCR analysis of cytoplasmic mtDNA content in TEPMs from HFD-fed Apoe -/ - mice with or without PEM.. n=10 per group. N , The ECAR and OCR in TEPMs from HFD-fed Apoe -/ - mice with or without PEM were monitored using a Seahorse XFe24 analyzer. n=6 per group. O , RT-qPCR analysis of inflammatory factors in plaque macrophages from HFD-fed Apoe -/ - mice with or without PEM. n=6 per group. Data represent the mean±SEM. * P <0.05, ** P <0.01, *** P <0.001 by 1-way ANOVA followed by Tukey multiple comparisons test ( B , D through F , and H ), unpaired 2-sided Student t test ( G , J , L , M , and O ), and unpaired 2-sided Student t test with Welch correction ( O ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Activity Assay, Purification, ATP Bioluminescent Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Expressing, Binding Assay, Staining, Dot Blot, Western Blot

Journal: Circulation

Article Title: METTL4-Mediated Mitochondrial DNA N6-Methyldeoxyadenosine Promoting Macrophage Inflammation and Atherosclerosis

doi: 10.1161/CIRCULATIONAHA.124.069574

Figure Lengend Snippet: PROTAC-PEM could effectively alleviate atherosclerosis. A , The preparation procedure for PROTAC-PEM targeting macrophages. B , Western blot analysis of METTL4 expression in HMDMs pretreated with MG132 (10 μM, 4 hours), followed by ox-LDL stimulation combining with PROTAC-PEM. n=6 per group. C , IP analysis of the binding of 6mA and MT-ATP6 in ox-LDL–stimulated HMDMs treated with or without PROTAC-PEM. n=10 per group. D , RT-qPCR (quantitative reverse transcription polymerase chain reaction) analysis of MT-ATP6 expression in ox-LDL–stimulated HMDMs treated with or without PROTAC-PEM. n=6 per group. E , ECAR and OCR in ox-LDL–stimulated HMDMs treated with or without PROTAC-PEM were monitored using a Seahorse XFe24 analyzer. n=6 per group. F and G , qPCR (quantitative polymerase chain reaction) analysis of cytoplasmic mtDNA content ( F ), RT-qPCR analysis of inflammatory factors ( G ) in ox-LDL–stimulated HMDMs treated with or without PROTAC-PEM. n=6 per group. H , En face Oil Red O staining of the aorta of NC or HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=6 per group. I , RT-qPCR analysis of CD11b in aortic root plaques derived from NC or HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=10 per group. J , RT-qPCR analysis of inflammatory factors in macrophages within atherosclerotic plaques of NC or HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=6 per group. K , Dot blot analysis of mtDNA 6mA levels in TEPMs from NC or HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=6 per group. L and M , RT-qPCR analysis of MT-ATP6 ( L ), and qPCR analysis of the cytoplasmic mtDNA content in TEPMs ( M ) from NC or HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=10 per group. N , Representative immunofluorescence staining images of macrophage (CD68, red) and IL-1β (green) in the aortic root derived from HFD-fed Apoe -/ - mice treated with or without PROTAC-PEM. n=6 per group. O , A mechanistic diagram of METTL4-mediated mtDNA 6mA modification in macrophage induced atherosclerosis development. Data represent the mean±SEM. * P <0.05, ** P <0.01, *** P <0.001 by 2-way ANOVA followed by Tukey multiple comparisons test ( B through D , F through J , and L and M ).

Article Snippet: Therefore, we performed homology modeling and molecular docking based on the crystal structure of the MT-A70 domain of Arabidopsis METTL4 and the human-derived METTL3 sequence to identify the compound (Figure A)., Using a structure-based drug discovery approach, we identified that xanthinol nicotinate, pemetrexed, and linagliptin, among the 18 compounds screened from TargetMol, could effectively inhibit the methyltransferase activity of METTL4 (Figure B and C).

Techniques: Western Blot, Expressing, Binding Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Staining, Derivative Assay, Dot Blot, Immunofluorescence, Modification