Journal: bioRxiv

Article Title: ChromSMF: integrated profiling of histone modifications, protein-DNA interactions and DNA methylation on multi-kilobase DNA molecules

doi: 10.64898/2026.03.11.710921

Figure Lengend Snippet: (A) Schematic representation of the two-step ChromSMF protocol . Cells are chemically permeabilized and incubated with M.CviPI (cytosine-MTase), a methyltransferase that deposits cytosine methylation at accessible GpCs across the genome (black spheres). The obligatory cofactor SAM is washed out to stop M.CviPI activity. Cells are then sequentially incubated with an antibody (Ab) targeting a histone modification, and the proteinA-Hia5 (adenine-MTase) fusion protein that will methylate adenines in proximity of the histone modification of interest (green spheres). Sequencing of the resulting DNA using Oxford Nanopore Technologies (ONT) enables simultaneous detection of histone modifications (green signal; mA) and transcription factor binding events (black signal; mC) at bulk and single-molecule resolution. (B) Example locus illustrating the simultaneous detection of H3K4me3 and chromatin accessibility at active promoters . Top panel: genome browser tracks displaying ChIP-seq enrichment for H3K4me3 (green) and DNase-seq signal (black). Lower panel: ChromSMF sample for H3K4me3. Average SMF signal (1 – mC%) of individual cytosines (black) and average smoothed mA signal (mA%; green; smoothing across 4 adenines). (C) Simultaneous detection of chromatin accessibility and H3K4me3 on individual DNA molecules at a locus with low ChIP-seq enrichment for H3K4me3 . Single-molecule stacks display either mA-H3K4me3 (green, left) or mC-chromatin accessibility (black, right) signal. Molecules are displayed in identical order in both panels and originate from the same sample. Single-molecule classification of H3K4me3 (green) and chromatin accessibility (black) are shown as stacked bar plots between the single-molecule stacks. Single-molecule quantification of total H3K4me3 and chromatin accessibility at the locus are shown at the bottom. (D) Simultaneous detection of chromatin accessibility and H3K4me3 on individual DNA molecules at a locus with high ChIP-seq enrichment for H3K4me3 . Single-molecule stacks display either mA-H3K4me3 (green, left) or mC-chromatin accessibility (black, right) signal. Molecules are displayed in identical order in both panels and originate from the same sample. Single-molecule classification of H3K4me3 (green) and chromatin accessibility (black) are shown as stacked bar plots between the single-molecule stacks. Single-molecule quantification of total H3K4me3 and chromatin accessibility at the locus are shown at the bottom.

Article Snippet: Fully methylated CpG and GpC gDNA (sample 4) was generated by two consecutive 30 min incubations at 37°C with 8 U/μg DNA of M.CviPI GpC methyltransferase (in-house production) supplemented with 1.2 mM SAM (NEB, B9003S), followed by one 30 min incubation at 37°C with 16 U/μg DNA of M.SssI CpG methyltransferase (NEB, M0226L) in the presence of 7.5 μM MgCl2 and 0.8 mM SAM (NEB, B9003S).

Techniques: Incubation, Methylation, Activity Assay, Modification, Sequencing, Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: METTL9 regulates N1-histidine methylation of zinc transporters to promote tumor growth

doi: 10.1101/2021.04.20.440582

Figure Lengend Snippet: (A, B, C) Knockout of Mettl9 in RM-1 tumor cells decreases cell growth. (A) Cell growth of wildtype (WT) and two different knockout (KO) clones of RM-1 cells was measured by CCK8 and (B, C) colony formation assay. (D-I) WT and Mettl9 KO RM-1 tumor cells were injected into nude mouse and C57B6/J mice. (D, G) Tumor growth curves in nude mice (n=6) and C57B6/J mice(n=5). (E, H) Pictures of tumors three weeks after tumor cell injection. (F, I) Tumor weights for (E, H). (J-N) Enzymatically active METTL9 methylates SLC39A7. (J) Fluorography showing the activity of recombinant METTL9 on cell extracts from WT and Mettl9 KO RM-1 cells in the presence of [ 3 H]AdoMet. Ponceau S staining for total protein was used as loading control (bottom). (K) Fluorography showing recombinant GST-SLC39A7 was methylated by recombinant METTL9 in the presence of [ 3 H]AdoMet. GST-P53 was used as a substrate control. (L, M) Fine mapping of the METTL9-methylated regions in SLC39A7. The methylated truncate was colored in red. (N) Fluorography showing in vitro activity of METTL9 on WT and mutated recombinant GST-A7(66-78). (O) Histidine (His, left), 3-(τ)-methyl histidine (His(3-me), center); 1-(π)-methyl histidine (His(1-me), right). (P) METTL9 generates His73(1-me). In vitro methylation reactions with GST-METTL9 on the indicated peptides visualized by autoradiography. (Q) Fluorography showing GHSH motif is adequate for METTL9 mediated methylation. In vitro methylation assay of METTL9 on recombinant GST-GHGHSH, GST-HGHSH and GST-GHSH. (R) In vitro activity of METTL9 on recombinant protein arrays. Residue in the GHSH motif (red) was replaced with 20 different amino acids. For all panels, *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Error bars represent S.D. Data are representative of three independent experiments.

Article Snippet: The primary antibodies against METTL9 (1:2000, ProteinTech) and β-actin (1:5000, ProteinTech) were used overnight at 4 °C, then washed the PVDF membranes three times with TBST (0.1% Tween-20 in TBS), the membranes was incubated with secondary antibody (1:5000, ProteinTech) for 40 minutes and washed three times with TBST.

Techniques: Knock-Out, Clone Assay, Colony Assay, Injection, Activity Assay, Recombinant, Staining, Control, Methylation, In Vitro, Autoradiography, Residue

Journal: bioRxiv

Article Title: METTL9 regulates N1-histidine methylation of zinc transporters to promote tumor growth

doi: 10.1101/2021.04.20.440582

Figure Lengend Snippet: (A) Fluorescence images of FluoZin-3 (2 μM, 1 h, 25 °C) stained WT RM-1 cell line (left), Mettl9 KO 1# cell line (middle), Mettl9 KO 2# cell line (right). Scale bar, 10 μm. (B) GO analysis of top pathways from differentially expressed genes in WT and Mettl9 KO RM-1 tumor cells. (C) The predicted structure model of SLC39A7. The number represents the peptide region in SLC39A7. Green color represents mutant residues. (D) In vitro activity of METTL9 on recombinant GST tagged WT and mutant peptides at the indicated region of SLC39A7. H204/216A, histidine to alanine mutation at 204 and 216 sites; H254/262A, histidine to alanine mutation at 254 and 262 sites. (E-H) Overexpression of WT and mutant SLC39A7 in RM-1 WT cells. (E, G) Fluorescence images of FluoZin-3 (2 μM, 1 h, 25 °C) stained the indicated groups of BFP-empty vector (EV), BFP-SLC39A7, BFP-SLC39A7 H204/216A and H254/262A mutants. Scale bar,10 μm. (F, H) Quantification of colony formation assays in (E, G) (see Methods). (I) METTL9 transcript levels were analyzed in cancer and normal tissues from the TCGA database combined with GTEx normal data. PRAD (Prostate adenocarcinoma) PAAD (Pancreatic adenocarcinoma) and LIHC (Liver hepatocellular carcinoma). (J, K) The overall survival rates of the METTL9 high-expression group and the METTL9 low-expression group in PAAD and LIHC cancer types from the TCGA data. (L) Heatmap correlation between several genes expression levels and immune scores calculated by ESTIMATE in tumor tissues among different TCGA cancer datasets. For all panels, *: P < 0.05; **: P < 0.01; ***: P < 0.001; unpaired two-tailed Student’s t-test. Error bars represent S.D. Data are representative of three independent experiments.

Article Snippet: The primary antibodies against METTL9 (1:2000, ProteinTech) and β-actin (1:5000, ProteinTech) were used overnight at 4 °C, then washed the PVDF membranes three times with TBST (0.1% Tween-20 in TBS), the membranes was incubated with secondary antibody (1:5000, ProteinTech) for 40 minutes and washed three times with TBST.

Techniques: Fluorescence, Staining, Mutagenesis, In Vitro, Activity Assay, Recombinant, Over Expression, Plasmid Preparation, Expressing, Two Tailed Test

Journal: International immunopharmacology

Article Title: The epigenetic-modified downregulation of LOXL1 protein mediates EMT in bladder epithelial cells exposed to benzo[a]pyrene and its metabolite BPDE.

doi: 10.1016/j.intimp.2024.113232

Figure Lengend Snippet: Fig. 5. DNMT3a and DNMT3b are key factors in the regulation of LOXL1 methylation modification. (A-D) The protein level of DNMT3a/3b was detected by WB assay in SV-HUC-1 cells exposed to the indicated concentrations (1 μM and 2 μM) of B[a]P/BPDE for 24 h (Fig. 5A), 1 month (Fig. 5B), 3 months (Fig. 5C), and 6 months (Fig. 5D); (E) DNMT3a knockdown construct was stably transfected into SV-HUC-1 cells, and knockdown efficiency was then determined using Western blot; (F) The efficiency of DNMT3a knockdown on downstream LOXL1 protein level was assessed using Western blot; (G) Stable transfection of DNMT3b knockdown plasmid into SV-HUC-1 cells and WB assay to detect its knockdown efficiency; (H) Western blot was used to detect the effect of DNMT3b knockdown on downstream protein LOXL1 expression.

Article Snippet: Antibodies specific against DNMT3a (sc-373905), DNMT3b (sc376043), LOXL1 (sc-166632), SLUG (sc-166476) were purchased from Santa Cruz Biotechnology (TX, USA); Antibodies specific against ECadherin (60335-1-Ig) and β-Actin (66009-1-Ig), were purchased from Proteintech (Wuhan, China).

Techniques: Methylation, Modification, Knockdown, Construct, Stable Transfection, Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: International immunopharmacology

Article Title: The epigenetic-modified downregulation of LOXL1 protein mediates EMT in bladder epithelial cells exposed to benzo[a]pyrene and its metabolite BPDE.

doi: 10.1016/j.intimp.2024.113232

Figure Lengend Snippet: Fig. 6. B[a]P/BPDE exposure induces DNMT3a and DNMT3b expression in SV-HUC-1 cells by generating ROS. (A) Intracellular ROS levels were assessed using the DCFH-DA probe; (B) Quantitative analysis of fluorescence intensity; (C) Detection of intracellular ROS levels after NAC (3 mM) treatment with the ROS inhibitor using DCFH-DA probe; (D) Quantitative analysis of fluorescence intensity; (E) WB assay was performed to detect the impact of inhibitor NAC on down stream proteins expression in SV-HUC-1 cells; (F) Cell scratch assay was used to detect the impact of ROS inhibitor NAC on the migration ability of SV-HUC-1 cells.

Article Snippet: Antibodies specific against DNMT3a (sc-373905), DNMT3b (sc376043), LOXL1 (sc-166632), SLUG (sc-166476) were purchased from Santa Cruz Biotechnology (TX, USA); Antibodies specific against ECadherin (60335-1-Ig) and β-Actin (66009-1-Ig), were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Fluorescence, Wound Healing Assay, Migration

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Epigenetically silenced lncRNA SNAI3-AS1 promotes ferroptosis in glioma via perturbing the m 6 A-dependent recognition of Nrf2 mRNA mediated by SND1.

doi: 10.1186/s13046-023-02684-3

Figure Lengend Snippet: Fig. 6 SND1 recognizes Nrf2 mRNA and enhances its stability in an m6A-dependent manner. A RIP assays were performed using anti-SND1 and IgG antibodies. The enrichments of Nrf2 mRNA by SND1 or IgG were detected via RT-qPCR. B After actinomycin D (5 μg/ml) treatment for 0, 2, 4, 6 h, RT-qPCR was used to analysis the Nrf2 mRNA stability in U87MG cells with SND1 overexpression and A172 cells with SND1 silence. C RT-qPCR detected the mRNA level of Nrf2 in U87MG cells with SND1 overexpression and A172 cells with SND1 silence. D Western blotting showed the protein levels of Nrf2 after SND1 overexpression or silence under (E) Schematic illustration was used to explain the design of dual luciferase reporter plasmids containing wild-type or mutant m6A sites in Nrf2 3’ UTR sequence. F Wild-type or mutant plasmids of reformed dual luciferase reporters were transfected into U87MG cells with SND1 overexpression and A172 cells with A172 cells with SND1 silence, respectively. The relative luciferase activity was measured and normalized. G m6A levels of U87MG cells with or without three m6A methyltransferases (METTL3, METTL14, and WTAP) silence were detected using the m6A RNA Methylation Quantification Kit and m6A dot blot assays. H U87MG cells with indicated interventions were treated with actinomycin D (5 μg/ml) for 0, 2, 4, 6 h, and Nrf2 mRNA stability was analyzed via RT-qPCR. I In METTL3-silenced or control U87MG cells, MeRIP assays and RT-qPCR were performed to calculate the relative enrichment of m6A modification. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and n.s., not significant

Article Snippet: The primary antibodies were listed as following: GAPDH (60,004–1-Ig, Proteintech), β-actin (66009–1- Ig, Proteintech), Nrf2 (16396–1-AP, Proteintech), SND1 (10760–1-AP, Proteintech), METTL3 (15073–1-AP, Proteintech), METTL14 (26158–1-AP, Proteintech), WTAP (10200–1-AP, Proteintech), FLAG (20543–1-AP, Proteintech), Keap1 (10503–2-AP, Proteintech), GPX4 (67763–1-Ig, Proteintech), 4-Hydroxynonenal (4-HNE, R&D systems, MAB3249).

Techniques: Quantitative RT-PCR, Over Expression, Western Blot, Luciferase, Mutagenesis, Sequencing, Transfection, Activity Assay, Methylation, Dot Blot, Control, Modification

Journal: Environmental Health Perspectives

Article Title: Immunotoxicity Evaluation of Trihalophenolic Disinfection By-Products in Mouse and Human Mononuclear Macrophage Systems: The Role of RNA Epitranscriptomic Modification in Mammalian Immunity

doi: 10.1289/EHP11329

Figure Lengend Snippet: The effects of TCP, TBP, and TIP on the expression of m 6 A methyltransferases, demethylases, and m 6 A binding proteins in RAW264.7 cells. (A–J) Transcriptional expression of m 6 A methyltransferases, demethylases, and m 6 A binding proteins in RAW264.7 cells. After exposure to different concentrations (0, 50, 100, 200 μ M ) of TCP, TBP, or TIP for 24 h, the mRNA levels of Mettl3 , Mettl14 , Wtap , Fto , Alkbh5 , Ythdf1 , Ythdf2 , Ythdf3 , Ythdc1 , and Ythdc2 were quantitatively measured via qRT-PCR. Results are shown as fold differences compared with the control group ( 0 μ M ). 1 = no difference . Data are expressed as mean ± SD ( n = 3 biological replicates, each with two technical replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. * p < 0.05 , ** p < 0.01 , *** p < 0.001 . The corresponding data are presented in Excel Table S7. (K,L) Protein abundance of METTL3, METTL14, and WTAP in RAW264.7 cells. After exposure to different concentrations (0, 50, 100, 200 μ M ) of TCP, TBP, or TIP for 24 h, protein abundance of METTL3, METTL14, WTAP, and the loading control (GAPDH) was analyzed using Western blotting. Western blotting was repeated with independent sample preparations three times ( n = 3 biological replicates; Figure S5). (K) One representative figure for each m 6 A methyltransferase was shown. (L) Western blotting results were quantified with Image Lab. 1 = no difference . Data are expressed as mean ± SD ( n = 3 biological replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. * p < 0.05 , ** p < 0.01 , *** p < 0.001 . The corresponding data are summarized in Excel Table S8. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; m 6 A , N 6 -methyladenosine ; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SD, standard deviation; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

Article Snippet: For Western blotting, N 6 -mA Methyltransferase Antibody Sampler Kit (1:1,000 dilution for METTL3, METTL14, and WTAP; Cat. #69159) and GAPDH Rabbit mAb (1:1,000 dilution; Cat. #2118) were bought from Cell Signaling Technology.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation