Fig 2: Downregulation of miR-509–3p transcription by COL11A1 increases DNMT1 phosphorylation and stability.(A) The mRNA expression levels of COL11A1 and miR-509–3p in A2780CP70 and OVCAR-8 cells transfected with miR-509–3p mimics and in A2780 and OVCAR-3 cells transfected with the miR-509–3p inhibitor were evaluated using real-time RT-PCR. All experiments were performed in triplicate. (B) Upper panel: mRNA expression levels of miR-509–3p and COL11A1 in A2780CP70 and OVCAR-8 cells transfected with COL11A1 shRNA were evaluated using real-time RT-PCR. All experiments were performed in triplicate. * P < 0.05, ** P < 0.01, compared with shV. Lower panel: mRNA expression levels of miR-509–3p and COL11A1 in A2780 and OVCAR-3 cells transfected with the COL11A1 cDNA plasmid were evaluated using real-time RT-PCR. All experiments were performed in triplicate. * P < 0.05, compared with V. (C) A2780 cells were transfected with a COL11A1 cDNA plasmid and A2780CP70 cells were transfected with COL11A1 shRNA. COL11A1, DNMT1, DNMT3A, DNMT3B were evaluated using western blotting. ß-actin was used as a loading control. (D) The binding activity of DNMT1, DNMT3A, and DNMT3B to the miR-509–3p promoter was evaluated using ChIP in COL11A1-overexpressing A2780 cells and COL11A1-knockdown A2780CP70 cells. Chromatin was isolated and immunoprecipitated using anti-DNMT1, -DNMT3A, and -DNMT3B antibodies, respectively. (E) The binding activity of DNMT1 to the miR-509–3p promoter was evaluated using ChIP in COL11A1-overexpressing A2780cells treated with 1 or 10 µM 5-aza-2'-deoxycytidine for 5 days. Chromatin was isolated and immunoprecipitated using an anti-DNMT1 antibody. (F) A2780 cells were transfected with a COL11A1 cDNA plasmid and A2780CP70 cells were transfected with COL11A1 shRNA. The COL11A1, Akt, p-Akt (ser473), DNMT1, DNMT1 (ser84), DNMT1 (ser154), and p16 levels were evaluated using western blotting. ß-actin was used as a loading control. (G) A2780 cells transfected with the COL11A1 cDNA plasmid and A2780CP70 cells transfected with COL11A1shRNA and the corresponding cell lysates were immunoprecipitated using anti-DNMT1 antibodies. The resulting immunoprecipitates (IPs) were analyzed via immunoblotting (IB) using an anti-ubiquitin antibody. (H) Representative bisulfite sequencing of A2780 cells transfected with the COL11A1 cDNA plasmid and of A2780CP70 cells transfected with COL11A1shRNA. (I) OVCAR-3 cells were transfected with a COL11A1 cDNA plasmid and OVCAR-8 cells were transfected with COL11A1 shRNA. The COL11A1, DNMT1, DNMT3A, DNMT3B, Akt, p-Akt (ser473), DNMT1 (ser84), DNMT1 (ser154), and p16 levels were evaluated using western blotting. ß-actin was used as a loading control. (J) OVCAR-3 cells were transfected with a COL11A1 cDNA plasmid and OVCAR-8 cells were transfected with COL11A1shRNA and the cell lysates were immunoprecipitated using anti-DNMT1 antibodies. The resulting IPs were analyzed via IB, using an anti-ubiquitin antibody. (K) The binding activity of DNMT1 to the miR-509–3p promoter was evaluated using ChIP in COL11A1-overexpressing OVCAR-3 cells and COL11A1-knockdown OVCAR-8 cells. Chromatin was isolated and immunoprecipitated using an anti-DNMT1 antibody. (L) Representative bisulfite sequencing of OVCAR-3 cells transfected with the COL11A1 cDNA plasmid and of OVCAR-8 cells transfected with COL11A1 shRNA.

Fig 3: The inhibitory effect of GA on DNMTs(A) Genomic 5 mC content of H1299, A549, and DOK cell lines with or without GA (10 µM) treatment for 120 or 240 h. Cells treated with 5azaC (10 µM) for 120 h were used as positive controls. Data are presented as means ± SD (n = 3). (B–C) The abundances of the nuclear (N) and cytoplasmic (C) DNMT1, DNMT3A, and DNMT3B in GA-treated H1299 were determined using western blotting. The amount of histone deacetylase 2 (HDAC2) or GAPDH detected on the same membrane (indicated by vertical lines) was used as a loading control for normalization. The intensity for each protein band was quantified using ImageJ. The relative fold-changes after normalization are presented. Normalized quantities of DNMTs at time zero were set as 100%.

Fig 4: CpG islands exist ubiquitously throughout the genome, often upstream of gene promoter elements. Methylation of CpG islands is typically associated with repression of downstream gene transcription. Cytosine residues in these CpG islands are often methylated by DNMT3A/3L and DNMT3B/3L complexes, whereas DNMT1 functions mainly to maintain 5-mC of hemi-methylated DNA during replication. DNMTs require the universal methyl donor SAM. DNA demethylation requires the coordinated action of TET enzymes with TDG and the base excision repair pathway, generating 5-hydroxymethylcytosine, (5-hmC), 5-fluorocytosine (5-fC), and 5-carboxylcytosine (5-caC) intermediates during the process. TETs require oxygen and a-ketoglutarate (aKG) as reaction substrates. Created with Biorender.com.

Fig 5: Effects of 24 h freezing, 8 h thaw, 24 h anoxia, and 40% dehydration on total protein levels of DNA methyltransferases DNMT1, DNMT3A, and DNMT3L in brain tissue of Rana sylvatica as determined by Western immunoblotting. Representative immunoblots are also shown, included with the approximate location of the closest molecular marker. DNMT3B antibodies did not cross-react strongly with protein homogenates in the target range. Data are mean ± SEM, on n = 4–5 independent trials on tissue samples from different animals. Values labeled with different letters (a, b) are significantly different from one another (p < 0.05). DNMT3A is n = 5 for frozen samples, while other targets are n = 4.