Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: (A) Schematic of generating human embryonic stem cell (hESC) models that harbor heterozygous growth syndrome-associated mutations in DNMT3A and NSD1 by CRISPR-Cas9 genome engineering. Schematic was created using BioRender. See for detailed genotypes of the mutant clones. (B) Relative expression of DNMT3A transcripts normalized to RNA18S transcript levels. Each dot represents an independent clone. (C) Western blot analysis of DNMT3A protein expression. DNMT3A knockout (KO) clones were included as controls. HDAC1 was used as a loading control. Each lane represents an independent clone. DNMT3A genotypes are as follows: WT, WT/WT; frameshift, WT/frameshift; R882H, WT/R882H; KO, frameshift/frameshift; GoF, WT/W330R or WT/D333N. The plots on the right show the relative intensity of DNMT3A bands, normalized to HDAC1. (D) Relative expression of NSD1 transcripts normalized to RNA18S transcript levels. (E) Mass spectrometry of histones H3.1 and H3.3 K36 modifications in WT and NSD1 LoF hESCs. Each dot represents an independent clone. unmod., unmodified; me1, monomethylated; me2, dimethylated; me3, trimethylated; ac, acetylated. For panels B, C, D, and E, Statistical significance was determined by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques: CRISPR, Mutagenesis, Clone Assay, Expressing, Western Blot, Knock-Out, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: Proportions of in each full-stack chromatin state for (A) DNMT3A LoF hESCs, (B) TBRS patient blood (HypoMPs n=832), (C) DNMT3A GoF hESCs, (D) a HESJAS patient peripheral blood leukocyte sample (HypoMPs n=2,796; HyperMPs n=9,576), (E) NSD1 LoF hESCs, and (F) Sotos syndrome patient blood (HypoMPs n=24,148; HyperMPs n=4,168) Background represents proportion of all probes in each state. HyperMPs of TBRS patients were not analyzed due to a small size (n=38). Statistical significance was determined by Fisher test. *, p<0.001.

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: (A) Number of overlapping HypoMPs between DNMT3A LoF and GoF mutants. P<2.2*10^-16, Fisher’s exact test of unique DNMT3A GoF in all probes compared to DNMT3A GoF shared with DNMT3A LoF. (B) Log2 odds ratios showing enrichment of shared HypoMPs from DNMT3A mutants across full-stack chromatin states. Top 10 enriched states are shown (all p<1*10-18). (C) Mean DNA methylation values at CpG positions within selected enhancer chromatin states. Each dot represents an independent clone. Statistical significance was determined by Student’s t-test. *, p<0.05; **, p<0.01; ***,p<0.001; ns, not significant. (D-E) RELI analysis of shared HypoMPs intersected with >10,000 public chromatin datasets. Shown are the top 200 datasets ranked by Z-score. Most enriched chromatin factors (D) and cell types (E) are highlighted. PSC TF: POU5F1, NANOG, SOX2, cohesion: RAD21, NIPBL, PRC1.1: BCOR, KDM2B, PCGF1, RYBP, RNF2. Controls represent randomly sampled EPIC probe regions. Each dot represents a dataset profiling a chromatin factor in a human cell type. Supplemental Table 2 contains RELI results of all examined datasets. PSC, pluripotent stem cells; TF, transcription factors. (F) CUT&RUN (H2AK119ub) or ChIP-seq (all others) signal in WT hESCs 10 kilobases upstream and downstream from the center of shared HypoMPs or matched control regions. BCOR, KDM2B, PCGF1, H3K36me2 occupancy data are from GEO accession number GSE104690, RNF2 data is from GSE105028, H3K36me3 is from ENCODE (ENCSR476KTK), and H2AK119ub is from GSE301386 (this study).

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques: DNA Methylation Assay, ChIP-sequencing, Control

Journal: International Journal of Molecular Sciences

Article Title: Estradiol-17β Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo

doi: 10.3390/ijms22073655

Figure Lengend Snippet: Luteal gene and protein expression of DNA methyltransferases (DNMT): DNMT1 ( A , B ); DNMT3A ( C , D ) and DNMT3B ( E , F ) in response to estradiol-17β (E2) treatment in vivo and in gilts on day 12 of the estrous cycle and pregnancy. Control—gilts receiving placebo infusions into both uterine horns. E2—gilts receiving infusions of either placebo into one randomly selected horn or E2 (833 ng or 33.3 µg/infusion) into the contralateral horn. As referential groups, gilts on day 12 of the estrous cycle (D12 Cyclic) and pregnancy (D12 Pregnant) were included. Values of DNMTs gene expression were normalized against geometrical mean of ACTB and PPIA expression values (luteal samples from in vivo model) or PPIA expression values (luteal samples from day 12 pregnant/cyclic pigs). Values of DNMTs protein expression were normalized against total protein content. Main effect of treatment was detected on luteal expression of DNMT1 mRNA and protein, DNMT3A protein, and DNMT3B mRNA and protein (E2-treated groups vs. the control group; p < 0.05). There was no significant effect of the site of hormone administration (i.e., the placebo-treated uterine horn vs. the hormone-treated uterine horn within the same animal) on luteal expression of DNMT1, DNMT3A and DNMT3B mRNA and protein. Data are expressed as the mean ± SEM. Different letters indicate statistically significant differences between the control- and hormone-treated gilts ( p < 0.05). Asterisk indicates statistical differences between gilts on day 12 of the estrous cycle and pregnancy ( p < 0.05).

Article Snippet: DNMT3A , N/A , DNMT3A (D23G1) Rabbit mAb , #3598 Cell Signaling Technology , Rabbit, monoclonal , 1:300.

Techniques: Expressing, In Vivo, Control, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Estradiol-17β Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo

doi: 10.3390/ijms22073655

Figure Lengend Snippet: List of antibodies used in Western blot analyses.

Article Snippet: DNMT3A , N/A , DNMT3A (D23G1) Rabbit mAb , #3598 Cell Signaling Technology , Rabbit, monoclonal , 1:300.

Techniques: Western Blot, Sequencing

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Structure of mouse DNMT3A isoforms and DNMT3L and positions of the nucleotide substitutions and resulting amino acid substitutions. The introduced nucleotides and resulting amino acids are shown in red. The ADD, PWWP, UDR, and methyltransferase (MTase) motifs of the catalytic domain are indicated by colored boxes. While both DNMT3A1 and DNMT3A2 are expressed in male and female germ cells, DNMT3A2 is the predominant form in FGOs. b Western blotting of DNMT3A ADD and DNMT3L ADD in wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] whole testes. This experiment was repeated twice independently. c Pie graph showing the genotypes of mice generated by crossing [ Dnmt3a ADD/+ , Dnmt3L ADD/+ ] males and females. A total of 597 mice were genotyped at 3 weeks old. The expected Mendelian ratios for wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] female and male are respectively 3.12%. d Boxplots comparing the body weights of wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] mice at 12 weeks old. Females and males were examined separately. All boxplots hereafter are defined as following: central bar, median; lower and upper box limits, 25th and 75th percentiles, respectively; whiskers, minimum and maximum value within the rage of (1st quartile − 1.5*(3rd quartile − 1st quartile)) to (3rd quartile + 1.5*(3rd quartile − 1st quartile)). ** p value = 2.0E-03 and *** p value = 4.4E−05 by two-tailed t-test. e Fertility of wild-type, Dnmt3a ADD/ADD , Dnmt3L ADD/ADD , and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] females and males that were crossed with a C57BL/6L partner. One to three stillborn pups were obtained from Dnmt3a ADD/ADD females per delivery, as indicated by a cross (†). ns 1 and ns 2 p value = 0.10 and 0.47, respectively, and ** p value = 4.92E−03 by two-tailed t -test. f Representative images of ovaries and testes obtained from wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] mice. Boxplots on the right show the size (long axis) of these reproductive tissues. ns p value = 0.61 and *** p value = 6.65E-05 by two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Western Blot, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Immunofluorescence staining of wild-type, Dnmt3L ADD/ADD , Dnmt3a ADD/ADD , and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] FGOs for DNMT3A (red) and DNMT3L (green). Nuclei were counterstained with DAPI. b Boxplots showing the relative fluorescence intensity of DNMT3A (top) and DNMT3L (bottom) in the nucleus of FGOs of indicated genotypes. The relative values were obtained by dividing the fluorescence intensity of the DAPI-dense region by that of the non-DAPI-dense region in the same nucleus. *** 1 – *** 6 p value = 2.25E-05, 3.10E−06, 5.86E−05, 1.38E-09, 2.23E−13, and 2.13E−13, respectively, by two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Scatter plots comparing CG methylation levels of 10-kb genomic bins between FGOs of the indicated genotypes. A comparison between wild-type and Dnmt3L knockout FGOs is also shown. Data from biological replicates were combined after confirming their consistency. b Genome browser view of CG methylation levels of 10-kb bins across a 10-Mb region in FGOs of the indicated genotypes. Violin plots on the right show distributions of CG methylation levels of 10-kb bins from the whole genome. The numbers on the right indicate the global CG methylation levels. c Scatter plots comparing CG methylation levels of 10-kb genomic bins between Dnmt3a ADD/ADD and Dnmt3L ADD/ADD FGOs. d CG methylation levels of the maternally and paternally methylated ICRs in FGOs of the indicated genotypes. e CG methylation levels of repeat elements (LINEs, LTRs, and SINEs) in FGOs of the indicated genotypes. The color code is as Fig. 3d. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Methylation, Comparison, Knock-Out

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Scatter plots comparing CG methylation levels of 10-kb bins between spermatozoa of the indicated genotypes. A comparison between wild-type and Dnmt3L knockout spermatogonia at postnatal day 10 (P10 SG) is also shown. Data from biological replicates were combined after confirming their consistency. While the wild-type data was produced from one sample, it was consistent with our previous dataset . b Genome browser view of CG methylation levels of 10-kb bins across a 10-Mb region (the same region as in Fig. ) in spermatozoa of the indicated genotypes. Violin plots on the right show distributions of CG methylation levels in 10-kb bins from the whole genome. The numbers on the right indicate the global CG methylation levels. c Scatter plots comparing CG methylation levels of 10-kb genomic bins between Dnmt3a ADD/ADD and Dnmt3L ADD/ADD spermatozoa. d CG methylation levels of the maternally and paternally methylated ICRs in spermatozoa of the indicated genotypes. e , CG methylation levels of repeat elements (LINEs, LTRs, and SINEs) in spermatozoa of the indicated genotypes. The color code is as Fig. 4d. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Methylation, Comparison, Knock-Out, Produced

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Changes in gene expression detected between wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] FGOs. Genes that are up-regulated and down-regulated in [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] FGOs are plotted in red and blue, respectively (fold-change > 2, FDR < 0.05). b Multi-dimensional scaling plots of the gene expression profiles of FGOs of the indicated genotypes. The profiles of the wild-type MII oocytes are also shown. Each color-coded dot shows single-cell data. c Representative images of embryos recovered at E9.5. Wild-type embryo (top) and embryo obtained from [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] oocyte fertilized with wild-type spermatozoa (bottom). This experiment was repeated twice independently. d Volcano plots showing gene expression differences of each parental allele between embryos obtained from wild-type and [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] oocytes. Data from the maternal (left) and paternal allele (right) are separately shown. Red dots indicate the maternally imprinted genes ( n = 35). P values by the exact test under a negative binomial distribution. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Gene Expression

Journal: Nature Communications

Article Title: Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

doi: 10.1038/s41467-024-47699-2

Figure Lengend Snippet: a Scatter plots comparing non-CG methylation levels of 10-kb genomic bins between FGOs of the indicated genotypes. A comparison between wild-type and Dnmt3L knockout FGOs is also shown. b Scatter plots comparing the non-CG methylation levels of 10-kb bins between spermatozoa of the indicated genotypes. A comparison between wild-type and Dnmt3L knockout P10 SG is also shown. c Genome browser view of CG methylation (mCG) and non-CG methylation (mCH) levels in FGOs and spermatozoa of the indicated genotypes. Two genomic regions of 4.6 Mb (left) and 4.0 Mb (right) are shown. H3K36me3 ChIP-seq peaks in wild-type FGOs and round spermatids (RS) are also shown. The 10-kb bins exhibiting non-CG methylation levels > 5% higher in [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] oocytes and spermatozoa compared to their wild-type counterparts are indicated in a row designated “ΔmCH>5%”. Four representative regions containing such high mCH bins are indicated by open boxes and their zoomed-in snapshots are displayed at the bottom. d Bar graphs showing the fractions of methylated cytosines in all cytosines for FGOs (top) and spermatozoa (bottom) of the indicated genotypes. Each bar is divided into subsegments representing mCH and mCG. e Pie charts showing the ratios of methylated CA, CT, and CC in FGOs (top) and spermatozoa (bottom) of the indicated genotypes. f A motif showing enrichment in high-mCH 1-kb bins of [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] FGOs and spermatozoa ( N = 1855) over randomly selected 1-kb bins. g Violin plots showing distributions of non-CG methylation levels of 1-kb bins in wild-type FGOs (left) and spermatozoa (right). The 1-kb bins exhibiting non-CG methylation levels > 20% higher in [ Dnmt3a ADD/ADD , Dnmt3L ADD/ADD ] compared to their wild-type counterparts were compared with randomly selected bins. *** 1 and *** 2 p value = 5.92E-291, and 3.44E−72, respectively, by two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: The FGOs were then incubated with primary antibodies against DNMT3A (NOVUS, 64B14446) or DNMT3L (Abcam, ab194094) (dilution 1:500) overnight at 4 °C.

Techniques: Methylation, Comparison, Knock-Out, ChIP-sequencing, Two Tailed Test

Journal: Redox Biology

Article Title: Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells

doi: 10.1016/j.redox.2020.101716

Figure Lengend Snippet: Hypermethylation of the SOD2 upstream regulatory region is dependent on DNMT3A. (A) Location and methylation status of six CpG sites in the SOD2 upstream regulatory region analysed by bisulphite sequencing (BSP) in control and DGCR8 knockdown hMSCs (n = 12 individual clones). Each circle represents a single sequencing reaction of each CpG site (white circles, unmethylated CpG sites; black circles, methylated CpGs). (B) Total ROS levels are measured by H 2 DCF-DA after 5-aza-dC treatment (2 μM) in DGCR8 knockdown hMSCs. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01). (C) Immunoblot showing the levels of SOD2 and Ras signalling components after 5-aza-dC treatment (2 μM) in DGCR8 knockdown hMSCs. (D) Mitochondrial membrane potential (MMP) was assessed by JC-1 staining in DGCR8 knockdown hMSCs. Representative fluorescence microscopy images of siGFP- or siDGCR8-transfected cells, as well as 5-aza-dC-treated DGCR8 knockdown cells. Scale bars, 200 μm. (E) The mRNA levels of DNMTs were measured by quantitative real-time PCR. Data were normalised to GAPDH mRNA levels. Error bars indicate the standard error of the mean of three independent experiments (* P < 0.05). (F) Western blot analysis of DNMT3A protein levels in DGCR8 knockdown hMSCs.

Article Snippet: After electrophoresis, proteins were transferred onto nitrocellulose membranes (GE Healthcare) and incubated overnight at 4 °C with appropriate primary antibodies: H-Ras rabbit polyclonal antibody (sc-520, Santa Cruz; 1:200), MEK1/2 (D1A5) rabbit monoclonal antibody (8727; Cell Signalling; 1:1000), phospho-Erk1/2 mouse monoclonal antibody (9106, Cell Signalling; 1:1000), Erk1/2 rabbit polyclonal antibody (9102, Cell Signalling; 1:1000), phospho-p53 (Ser15) rabbit antibody (9284, Cell Signalling; 1:500), p53 rabbit polyclonal antibody (sc-6243, Santa Cruz; 1:1000), p21 rabbit monoclonal antibody (2947, Cell Signalling; 1:1000), SOD1 rabbit polyclonal antibody (sc-11407, Santa Cruz; 1:1000), SOD2 mouse monoclonal antibody (611,580, BD Biosciences; 1:1000), SOD3 mouse monoclonal antibody (ab28442, Abcam; 1:2000), DNMT3A rabbit polyclonal antibody (2160, Cell Signalling; 1:1000), alpha-tubulin rabbit antibody (SAB3501071, Sigma Aldrich; 1:5000), and alpha-tubulin mouse antibody (T9026, Sigma Aldrich; 1:5000).

Techniques: Methylation, Bisulfite Sequencing, Control, Knockdown, Clone Assay, Sequencing, Western Blot, Membrane, Staining, Fluorescence, Microscopy, Transfection, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells

doi: 10.1016/j.redox.2020.101716

Figure Lengend Snippet: miR-29a-3p and miR-30c-5p directly repress DNMT3A and rescue oxidative stress and premature senescence induced by DGCR8 knockdown. (A) Schematic illustration of miR-30c-5p (site 1) and miR-29a-3p (site 2) binding sites within the human DNMT3A 3′ UTR (662–868) region, which was cloned downstream of the Renilla luciferase reporter gene in the psiCHECK-2 vector. Two predicted targeting sites and the corresponding mutations are listed. The mutagenised nucleotides are underlined and highlighted in red. The construct for the mutant 3′ UTR contained both mutagenised sites (M1+M2). Data were normalised to firefly luciferase. (B) Luciferase reporter assay. 293T cells were co-transfected with luciferase reporters carrying either the wild-type DNMT3A 3′ UTR (Dnmt3a) or the mutagenised DNMT3A 3′ UTR (mDnmt3a), as well as 50 nM negative control mimic (miR-Control) or miR-29a-3p or/and miR-30c-5p mimics. Error bars indicate the standard error of the mean of three experiments. (** P < 0.01 and *** P < 0.001 compared to the miR-control). (C) The mRNA and protein expression levels of DNMT3A in DGCR8 knockdown hMSCs with or without miR-29a-3p or/and miR-30c-5p overexpression. The mRNA levels of DNMT3A were normalised to GAPDH . Error bars denote the standard error of the mean of three independent experiments (top). Immunoblot showing DNMT3A protein levels in DGCR8 knockdown cells with or without miR-29a-3p or/and miR-30c-5p forced expression (bottom). (D) Total ROS levels in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression were measured using H 2 DCF-DA. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01). Immunoblot showing the protein levels of SOD2 and Ras signalling components (bottom). (E) Mitochondrial membrane potential (MMP) in DGCR8 knockdown hMSCs after miR-29a-3p or/and miR-30c-5p overexpression was determined using JC-1. Representative fluorescence microscopy images in siGFP, siDGCR8, and mimic-transfected siDGCR8 hMSCs. Scale bar, 100 μm. (F) Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53 (ser15), after miR-29a-3p or/and miR-30c-5p overexpression in DGCR8 knockdown hMSCs. (G) Mean growth rate of siGFP and siDGCR8 hMSCs co-transfected with miR-29a-3p or/and miR-30c-5p mimics, as determined by MTS. Error bars denote the standard error of the mean of three independent experiments (** P < 0.01).

Article Snippet: After electrophoresis, proteins were transferred onto nitrocellulose membranes (GE Healthcare) and incubated overnight at 4 °C with appropriate primary antibodies: H-Ras rabbit polyclonal antibody (sc-520, Santa Cruz; 1:200), MEK1/2 (D1A5) rabbit monoclonal antibody (8727; Cell Signalling; 1:1000), phospho-Erk1/2 mouse monoclonal antibody (9106, Cell Signalling; 1:1000), Erk1/2 rabbit polyclonal antibody (9102, Cell Signalling; 1:1000), phospho-p53 (Ser15) rabbit antibody (9284, Cell Signalling; 1:500), p53 rabbit polyclonal antibody (sc-6243, Santa Cruz; 1:1000), p21 rabbit monoclonal antibody (2947, Cell Signalling; 1:1000), SOD1 rabbit polyclonal antibody (sc-11407, Santa Cruz; 1:1000), SOD2 mouse monoclonal antibody (611,580, BD Biosciences; 1:1000), SOD3 mouse monoclonal antibody (ab28442, Abcam; 1:2000), DNMT3A rabbit polyclonal antibody (2160, Cell Signalling; 1:1000), alpha-tubulin rabbit antibody (SAB3501071, Sigma Aldrich; 1:5000), and alpha-tubulin mouse antibody (T9026, Sigma Aldrich; 1:5000).

Techniques: Knockdown, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, Mutagenesis, Reporter Assay, Transfection, Negative Control, Control, Expressing, Over Expression, Western Blot, Membrane, Fluorescence, Microscopy

Journal: Cells

Article Title: Zscan4 Contributes to Telomere Maintenance in Telomerase-Deficient Late Generation Mouse ESCs and Human ALT Cancer Cells

doi: 10.3390/cells11030456

Figure Lengend Snippet: Reduction of DNA methylation and H3K9me3 occupancy at Zscan4 promoter regions in G4 Terc -/- ES cells with short telomeres. ( A ) Relative quantity (RQ) of DNA methylation levels among WT, G4 Terc -/- , and 5-aza-dC treated WT ES cells (1 µM for 2.5 days). Quantification was made by quantifying mean fluorescence intensity of cell populations following immunostaining with anti-5mC and 5hmC antibodies and analyzed by FlowJo software. Error bars indicate mean ± SEM ( n = 3). ***, p < 0.001, compared to WT controls. ( B ) Western blotting shows decreased Dnmt1, Dnmt3a, Dnmt3b, and increased Tet2 protein levels in G4 Terc -/- ES cells, respectively. Two technical repeats were included. ( C ) Western blotting shows decreased Dnmt1, Dnmt3b protein levels after 5-aza-dC treatment (1 µM for 2.5 days) in WT ES cells. ( D ) Bisulfate sequencing analysis of CpG dinucleotides of mouse Zscan4c proximal promoter regions among WT, G4 Terc -/- and 5-aza-dC treated WT ES cells (1 µM for 2.5 days). Open circles, unmethylated; closed circles, methylated. ( E ) Quantification of bisulfate genomic sequencing results of 10–11 independent clones (n) at Zscan4c proximal promoter. Note the decrease in methylated CpGs at Zscan4c proximal promoter in G4 Terc -/- and 5-aza-dC treated WT ES cells compared with WT ES cells. Error bars indicate mean ± SEM. ( F ) Representative images shows immunofluorescence staining of heterochromatic H3K9me3 (red). Nuclei stained with Hoechst33342 (blue) show brighter fluorescence signal in the heterochromatin. Scale bar, 10 μm. ( G ) Quantification of relative H3K9me3 mean fluorescence intensity between WT and G4 Terc -/- ES cells using ImageJ software. Randomly chosen cells from 9–10 fields of view were counted. n, number of cells counted. ( H ) Western blotting analysis shows the decreased inhibitive histone modification H3K9me3 in G4 Terc -/- ES cells. ( I ) ChIP-qPCR analysis of H3K9me3 occupancy at six proximal locations upstream to Zscan4c promoter, sub-telomere further upstream to Zscan4c promoter of chr7 and Tcstv1 / Tcstv3 promoter at subtelomere of chr13. Relative fold enrichment was normalized to rabbit IgG ChIP signals at the same regions, and the β-actin locus served as negative controls. Error bars indicate mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to controls.

Article Snippet: Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 1–2 h. Blots were then probed with various primary antibodies, anti-Zscan4 (AB4340, Millipore, 1:1000), Dnmt1 (sc10221, Santa Cruz, 1:500), Dnmt3a (ab13888, Abcam, 1:1000), Dnmt3b (ab13604, Abcam, 1:1000), Tet2 (Kind gift from Dr. Jinsong Li from SIBS, 1:1000), H3K9me3 (ab8898, Abcam, 1:2000), H3 (ab1791, Abcam, 1:2000), and β-actin ((P30002, Abmart, 1:5000) by overnight incubation in 5% skim milk in TBST at 4 °C.

Techniques: DNA Methylation Assay, Fluorescence, Immunostaining, Software, Western Blot, Sequencing, Methylation, Genomic Sequencing, Clone Assay, Immunofluorescence, Staining, Modification

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 3. DNMT3a upregulation is responsible for the hypermethylation of the miR-200c gene promoter. (A) Knockdown of DNMT3a protein in AGS cells following 0, 24 and 48 h of transfection with 100 nM siRNA relative to negative controls. (B) Enhanced expression of DNMT3a protein in the MGC803 cells after stable transfection with the DNMT3a expression vector. (C and D) Bisulfite sequencing PCR showed that DNMT3a knockdown decreased the methyla- tion status of the miR-200c promoter region in the AGS cells, and enhanced DNMT3a expression increased the methylation status of the miR‑200c promoter region in the MGC803 cells. (E) Knockdown of DNMT3a resulted in upregulation of miR-200c expression, whereas enhanced DNMT3a expression resulted in decreased miR-200c expression (*P<0.05, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Knockdown, Transfection, Expressing, Stable Transfection, Plasmid Preparation, Methylation Sequencing, Methylation

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 2. Differential expression of DNMT3a in cell lines and tissues. (A) Western blotting showed that the expression of DNMT3a in five GC cell lines was higher than that in immortal gastric epithelial GES-1 cells. (B) Immunohistochemistry assays showed that the expression of DNMT3a in GC tissues was significantly higher compared to that noted in the adjacent non-tumor tissues (magnification, x200).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Immunohistochemistry

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 4. miR-200c suppresses DNMT3a expression and induces endogenous pre-miR-200c and pri-miR-200c reexpression. (A) Putative miR-200c binding sites within the human DNMT3a 3'UTR are shown. The position of the binding sites was numbered relative to the first nucleotide of the 3'UTR. Mutations were introduced into DNMT3a 3'UTR that matched the seed region of miR-200c. (B) Interspecies conservation of putative miR-200c binding sites within the DNMT3a 3'UTR are shown. (C) Transfection with miR-200c reduced DNMT3A protein expression, whereas transfection with miR-200c inhibitors increased DNMT3A protein expression in the SGC7901 (left) and AGS cells (right). (D and E) Relative expression of pre-miR-200c and pri-miR-200c demonstrated by qRT-PCR was increased in the AGS cells following 0, 24 and 48 h transfection with 50 nM miR-200c mimics. (F) Ectopic miR-200c expression inhibits wild-type but not mutant DNMT3A 3'UTR reporter activity in the SGC7901 cells. Cells were co-transfected with miR-200c mimics and with either wild-type or mutant DNMT3A 3'UTR reporter construct. Luciferase activity assay was performed at 48 h after transfection (*P<0.05, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Expressing, Binding Assay, Transfection, Quantitative RT-PCR, Mutagenesis, Activity Assay, Construct, Luciferase

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 5. miR-200c mimics and DNMT3a siRNA suppress cell growth. (A and B) SGC7901 and AGS cells transfected with miR-200c mimics showed significantly reduced cellular proliferation compared with the negative control using Cell Counting Kit-8 assay. (C and D) SGC7901 and AGS cells transfected with DNMT3a siRNA also showed significantly reduced cellular proliferation compared with the negative control assaying (*P<0.05).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Transfection, Negative Control, Cell Counting

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 6. miR-200c mimics and DNMT3a siRNA suppress cell migration and invasion. (A) Representative images of cell invasion assays showed that miR- 200c mimics suppressed cell invasion in the SGC7901 and AGS cells. (B) Statistical analyses showed that miR-200c mimics suppressed cell invasion in the SGC7901 and AGS cells. (C) Representative images of cell invasion assays showed that DNMT3a siRNA suppressed cell invasion in the SGC7901 and AGS cells. (D) Statistical analyses showed that DNMT3a siRNA suppressed cell invasion in the SGC7901 and AGS cells. (E) Representative images of wound- healing assays showed that miR-200c mimics suppressed cell migration in the SGC7901 and AGS cells. (F) Statistical analyses showed that miR-200c mimics suppressed cell migration in the SGC7901 and AGS cells. (G) Representative images of wound-healing assays showed that DNMT3a siRNA suppressed cell migration in the SGC7901 and AGS cells. (H) Statistical analyses showed that DNMT3a siRNA suppressed cell migration in the SGC7901 and AGS cells (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Migration

Journal: Journal of Biological Chemistry

Article Title: Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci

doi: 10.1074/jbc.m110.170704

Figure Lengend Snippet: FIGURE 1. Dnmt3a2 expression and DNA methylation are reduced in Gsk-3 DKO ESCs. A, Western blot of Dnmt3a isoforms in WT and Gsk-3 DKO ESCs. Protein expression of Gsk-3 isoforms in WT and Gsk-3 DKO ESCs is shown in the middle panel, whereas a Western blot for tubulin, which serves as a loading control, is shown in the bottom panel. B, real-time quantitative PCR of Dnmt3a2 showing mRNA expression of Dnmt3a2 in Gsk-3 DKO ESCs relative to WT ESCs (RQ relative quantitation). Error bars represent S.D. between biological replicates, n 3 (***, p 0.001, two-tailed t test). C, real-time quantitative PCR showing H19 and Igf2 mRNA expression in Gsk-3 DKO ESCs relative to WT ESCs. Error bars represent S.D. between biological replicates, n 3 (*, p 0.05; ***, p 0.001, two-tailed t test). D, bisulfite sequencing analysis of the Igf2/H19 DMD. Circles represent 15 CpG dinucleotides analyzed within the Igf2/H19 DMD. Open circles represent unmethylated CpG dinucleotides, whereas filled circles represent methylated CpG dinucleotides (p 0.01, two-tailed t test).

Article Snippet: Antitubulin mouse mAb clone B-5-1-2 (Sigma) was diluted 1:10,000; anti-Dnmt3a mouse mAb clone 64B1446 (IMGENEX) was diluted 1:250; and anti-GSK-3 / mouse mAb clone 1H8 (Calbiochem), anti-phospho-GSK-3 / (Cell Signaling antibody 9331), anti-N-Myc (Cell Signaling antibody 9405), anti-c-Myc D84C12 XP (Cell Signaling antibody 5605), anti-PI3kinase p110 subunit C73F8 (Cell Signaling antibody 4249), anti-Akt1 2H10 (Cell Signaling antibody 2967), and anti-phospho-Akt Ser-473 193H12 (Cell Signaling antibody 4058) were all used at a 1:1000 dilution.

Techniques: Expressing, DNA Methylation Assay, Western Blot, Control, Real-time Polymerase Chain Reaction, Quantitation Assay, Two Tailed Test, Methylation Sequencing, Methylation