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: 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: Developmental biology

Article Title: Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

doi: 10.1016/j.ydbio.2006.04.436

Figure Lengend Snippet: Fig. 1. Expression dynamics of DNA methyltransferases during male germ cell development. Relative quantification of (A) Dnmt1, (B) Dnmt3a, and (C) Dnmt3b expression in isolated populations of male germ cells. Real-time RT- PCR was used to determine the expression levels of the three Dnmt genes in total RNA extracted from type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression of each gene was determined in triplicate in each of the two series of germ cells; shown here are the mean expression results obtained for one series. Mean ± SD.

Article Snippet: Membranes were blocked in 5% non-fat dried milk and were incubated with one of the following primary antibodies diluted in blocking buffer: clone 64B1446, a monoclonal antibody (mAb) raised against recombinant mouse DNMT3a (1:400; Imgenex, San Diego, CA); NB 100– 265, a polyclonal antibody (pAb) raised against amino acids 10–118 of human DNMT3A (1:2000; Novus Biologicals, Littleton, CO); clone 52A1018, a mAb raised against recombinant mouse DNMT3b (1:800; Imgenex); the specificity of these antibodies had previously been assessed by Chen et al. (2002), Beaulieu et al. (2002) and Weisenberger et al. (2004), respectively.

Techniques: Expressing, Quantitative Proteomics, Isolation, Quantitative RT-PCR

Journal: Developmental biology

Article Title: Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

doi: 10.1016/j.ydbio.2006.04.436

Figure Lengend Snippet: Fig. 2. Differential expression of Dnmt3a and Dnmt3a2 in male germ cells. (A) Structure of the mouse Dnmt3a locus (top) and the two main mRNA species produced from it (bottom). The Dnmt3a2-unique exon is marked by an asterisk. Exons are shown as black bars (exon sizes and positions are not to scale; adapted from Chen et al., 2002). (B) Relative expression of Dnmt3a (top) and Dnmt3a2 (bottom) in purified populations of male germ cells. QRT-PCR was used to determine the expression levels of the two transcripts in total RNA extracted from type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression of each transcript was determined in triplicate in each of the two series of germ cells; shown here are the mean expression results obtained for one series. Mean ± SD.

Article Snippet: Membranes were blocked in 5% non-fat dried milk and were incubated with one of the following primary antibodies diluted in blocking buffer: clone 64B1446, a monoclonal antibody (mAb) raised against recombinant mouse DNMT3a (1:400; Imgenex, San Diego, CA); NB 100– 265, a polyclonal antibody (pAb) raised against amino acids 10–118 of human DNMT3A (1:2000; Novus Biologicals, Littleton, CO); clone 52A1018, a mAb raised against recombinant mouse DNMT3b (1:800; Imgenex); the specificity of these antibodies had previously been assessed by Chen et al. (2002), Beaulieu et al. (2002) and Weisenberger et al. (2004), respectively.

Techniques: Quantitative Proteomics, Produced, Expressing, Purification, Quantitative RT-PCR

Journal: Developmental biology

Article Title: Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

doi: 10.1016/j.ydbio.2006.04.436

Figure Lengend Snippet: Fig. 3. Expression of Dnmt3aα and Dnmt3aβ in mouse germ cells. (A) Schematic representation of the 5′ region of the mouse Dnmt3a gene. The shaded area enlarged below shows an additional first exon (exon 1β) situated 5′ of the first exon that was initially identified (exon 1α). Splicing of exon 1α to exon 2 produces the Dnmt3aα transcript, while splicing of exon 1β to exon 2 gives rise to the Dnmt3aβ transcript (both splicing events are depicted here). Exons are represented as black bars (adapted from Weisenberger et al., 2002). (B) Expression of both Dnmt3aα and Dnmt3aβ in isolated male germ cells. RT-PCR was used to establish the incidence of transcription through exons 1α and 1β in total RNA extracted from type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). −RT, negative control lacking reverse transcriptase.

Article Snippet: Membranes were blocked in 5% non-fat dried milk and were incubated with one of the following primary antibodies diluted in blocking buffer: clone 64B1446, a monoclonal antibody (mAb) raised against recombinant mouse DNMT3a (1:400; Imgenex, San Diego, CA); NB 100– 265, a polyclonal antibody (pAb) raised against amino acids 10–118 of human DNMT3A (1:2000; Novus Biologicals, Littleton, CO); clone 52A1018, a mAb raised against recombinant mouse DNMT3b (1:800; Imgenex); the specificity of these antibodies had previously been assessed by Chen et al. (2002), Beaulieu et al. (2002) and Weisenberger et al. (2004), respectively.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Reverse Transcription

Journal: Developmental biology

Article Title: Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

doi: 10.1016/j.ydbio.2006.04.436

Figure Lengend Snippet: Fig. 5. Immunoblot detection of DNMT3a and DNMT3b in purified populations of male germ cells. (A) Schematic representation of DNMT3a and DNMT3a2. The conserved PWWP and PHD domains and methyltransferase motifs (I, IV, VI, IX, X) are depicted. The location of the different epitopes is underlined. (B) Expression of DNMT3a and DNMT3a2 in male germ cells. Top panel: detection of DNMT3a in protein lysates obtained from type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB) using the NB 100–265 antibody. Middle panel: expression of DNMT3a and DNMT3a2 in the same germ cell populations using the 64B1446 antibody. Bottom panel: gel electrophoresed under identical conditions but stained with Coomassie Brilliant blue dye demonstrating equal protein loading in each lane. (C) Detection of multiple DNMT3b isoforms in male germ cells. Upper panel: Western blot showing the presence of several DNMT3b isoforms in total protein extracted from various populations of male germ cells and from 6 (D6)- and 70 (D70)-dpp testes using the 52A1018 antibody (lane abbreviations are the same as in B for germ cells). Lower panel: gel electrophoresed under identical conditions but stained with Coomassie Brilliant blue dye demonstrating equal protein loading in each lane.

Article Snippet: Membranes were blocked in 5% non-fat dried milk and were incubated with one of the following primary antibodies diluted in blocking buffer: clone 64B1446, a monoclonal antibody (mAb) raised against recombinant mouse DNMT3a (1:400; Imgenex, San Diego, CA); NB 100– 265, a polyclonal antibody (pAb) raised against amino acids 10–118 of human DNMT3A (1:2000; Novus Biologicals, Littleton, CO); clone 52A1018, a mAb raised against recombinant mouse DNMT3b (1:800; Imgenex); the specificity of these antibodies had previously been assessed by Chen et al. (2002), Beaulieu et al. (2002) and Weisenberger et al. (2004), respectively.

Techniques: Western Blot, Purification, Expressing, Staining

Journal: Developmental biology

Article Title: Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

doi: 10.1016/j.ydbio.2006.04.436

Figure Lengend Snippet: Fig. 6. Schematic representation of DNA methyltransferase dynamics during spermatogenesis. Progression of methylation pattern establishment and maintenance, and expression of Dnmt1 (black), Dnmt3a (red), Dnmt3a2 (blue) and Dnmt3b (green) are presented as functions of male germ cell development. The intensity of the blue shading in germ cells reflects the methylation status of the paternal genome. Some of the most characteristic biological processes associated with mitosis, meiosis, and spermiogenesis are also depicted. Representative cell types are indicated as follows: A, type A spermatogonia; B, type B spermatogonia; PL, preleptotene spermatocyte, L/Z, leptotene/zygotene spermatocyte; PP, prepubertal pachytene spermatocyte; P, pachytene spermatocyte; RS, round spermatid; ES, elongating spermatids. MI–MII, meiosis I–II.

Article Snippet: Membranes were blocked in 5% non-fat dried milk and were incubated with one of the following primary antibodies diluted in blocking buffer: clone 64B1446, a monoclonal antibody (mAb) raised against recombinant mouse DNMT3a (1:400; Imgenex, San Diego, CA); NB 100– 265, a polyclonal antibody (pAb) raised against amino acids 10–118 of human DNMT3A (1:2000; Novus Biologicals, Littleton, CO); clone 52A1018, a mAb raised against recombinant mouse DNMT3b (1:800; Imgenex); the specificity of these antibodies had previously been assessed by Chen et al. (2002), Beaulieu et al. (2002) and Weisenberger et al. (2004), respectively.

Techniques: Methylation, Expressing

Journal: British Journal of Cancer

Article Title: MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

doi: 10.1038/sj.bjc.6605195

Figure Lengend Snippet: In silico prediction of the miR-143 target. ( A ) Predicted binding of miR-143 (grey) to DNMT3A 3′UTR (black). ( B ) Sequence alignment of human miR-143 with different species of DNMT3A 3′UTR. The seed sequence of miR-143 (bold) perfectly matches with the 3′UTR of DNMT3A.

Article Snippet: The membranes were probed with 1 : 1000 diluted primary antibodies against DNMT3A (Epigentek, Brooklyn, NY, USA) at room temperature for 2 h, washed extensively with 0.1% Tween-20 in PBS, and incubated with secondary antibodies conjugated with horseradish peroxidase (1 : 10 000 dilution).

Techniques: In Silico, Binding Assay, Sequencing

Journal: British Journal of Cancer

Article Title: MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

doi: 10.1038/sj.bjc.6605195

Figure Lengend Snippet: The functional effect of ectopic miR-143 expression in 228 and SW480 cells. ( A ) Ectopic miR-143 expression suppresses cell proliferation in colon cancer cells. Cells were transfected with miR-143 precursor or control precursor for 24 h. Cell proliferation at 5 days after transfection was assessed by MTT assay. The miR-143 expression was examined by qPCR (Mann–Whitney test, * P <0.05, ** P <0.01). Relative cell proliferation was compared with the corresponding control inhibitor or precursor (Mann–Whitney test, * P <0.05). ( B ) Ectopic miR-143 expression reduced the anchorage-independent growth of cancer cells using soft-agar colony formation assay. Cells were plated in 0.3% noble agar for 3 weeks. The number of colonies was counted after staining with 0.05% crystal violet (Mann–Whitney test, * P <0.05). Ectopic miR-143 expression reduced both ( C ) mRNA and ( D ) protein expression of DNMT3A. Cells were transfected with miR-143 precursor or with control precursor for 24 h, and then lysed for RNA or protein extraction. DNMT3A mRNA was detected by real-time qPCR (Mann–Whitney test, * P <0.05, ** P <0.01) and protein expression was detected by western blotting with an anti-DNMT3A antibody. β -Actin was used as a loading control.

Article Snippet: The membranes were probed with 1 : 1000 diluted primary antibodies against DNMT3A (Epigentek, Brooklyn, NY, USA) at room temperature for 2 h, washed extensively with 0.1% Tween-20 in PBS, and incubated with secondary antibodies conjugated with horseradish peroxidase (1 : 10 000 dilution).

Techniques: Functional Assay, Expressing, Transfection, Control, MTT Assay, MANN-WHITNEY, Soft Agar Assay, Staining, Protein Extraction, Western Blot

Journal: British Journal of Cancer

Article Title: MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

doi: 10.1038/sj.bjc.6605195

Figure Lengend Snippet: DNMT3A is the direct target of miRNA-143. ( A ) The wild-type (WT) and mutated (MUT) 3′UTR of DNMT3A, with the seed region (bold) and base substitutions (bold and italic), were sub-cloned into luciferase reporter construct and are shown. ( B ) Ectopic miR-143 expression inhibits wild-type but not mutant DNMT3A 3′UTR reporter activity in 228 and SW480 cells. Cells were co-transfected with miR-143 precursor and with either WT or MUT DNMT3A 3′UTR reporter construct. Luciferase activity assay was performed at 24 h after transfection (Mann–Whitney test, * P <0.05, ** P <0.01).

Article Snippet: The membranes were probed with 1 : 1000 diluted primary antibodies against DNMT3A (Epigentek, Brooklyn, NY, USA) at room temperature for 2 h, washed extensively with 0.1% Tween-20 in PBS, and incubated with secondary antibodies conjugated with horseradish peroxidase (1 : 10 000 dilution).

Techniques: Clone Assay, Luciferase, Construct, Expressing, Mutagenesis, Activity Assay, Transfection, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

doi: 10.1038/sj.bjc.6605195

Figure Lengend Snippet: Relationship between miR-143 and DNMT3A. Knockdown of DNMT3A inhibited cell proliferation in 228 and SW480 cells. Cells were transfected with DNMT3A siRNA for 24 h. Transfection with scramble siRNA was used as negative control. ( A ) The DNMT3A protein level 5 days after transfection was examined by western blot. The experiment has been repeated thrice. ( B ) Cell proliferation 5 days after transfection was assessed using MTT assay. Relative cell proliferation was compared with the corresponding scramble siRNA transfection by the Mann–Whitney test, * P <0.01. ( C ) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in seven colon cancer cell lines. Inverse correlation was obtained using Spearman's correlation, r =−0.78, P <0.05. ( D ) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in 10 paired adjacent normal and CRC tissues. Inverse correlation was also obtained by Spearman's correlation, r =−0.59, P <0.01. ( E ) The DNMT3A protein level was semi-quantified using western blot analysis of CRC tumours (T) and of adjacent normal cells (N). ( F ) The scatter plot of the fold changes of the miR-143 and DNMT3A protein (Log 10 scale at both X axis and Y axis) in seven paired CRC samples (Spearman's correlation, r =0.71, P <0.05).

Article Snippet: The membranes were probed with 1 : 1000 diluted primary antibodies against DNMT3A (Epigentek, Brooklyn, NY, USA) at room temperature for 2 h, washed extensively with 0.1% Tween-20 in PBS, and incubated with secondary antibodies conjugated with horseradish peroxidase (1 : 10 000 dilution).

Techniques: Knockdown, Transfection, Negative Control, Western Blot, MTT Assay, MANN-WHITNEY, Expressing

Journal: 3 Biotech

Article Title: Gandouling protects against hepatic fibrosis in Wilson disease through the lncRNA-SNHG7/miR-29b/DNMT3A pathway

doi: 10.1007/s13205-026-04769-0

Figure Lengend Snippet: GDL regulates the SNHG7/miR-29b/DNMT3A axis in vivo and in vitro . A Expression of SNHG7, miR-29b, and DNMT3A in liver tissues ( n = 6). B Expression of SNHG7, miR-29b, and DNMT3A in LX-2 cells ( n = 6). C Protein levels of DNMT3A and p-DNMT3A in liver tissues ( n = 3). D Protein levels of DNMT3A and p-DNMT3A in LX-2 cells ( n = 3). GAPDH was used as a loading control. Data are presented as means ± SD. ## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. model group; ** p < 0.01 vs. CuSO 4 group

Article Snippet: The membranes were then incubated overnight with antibodies against DNMT3A (Bioss, Item No. bs-23029R, 1:2000, Rabbit, 1:1000, Rabbit), p-DNMT3A (Bioss, Item No. bs-14399R, 1:2000, Rabbit), α-SMA (Bioss, Item No. Bsm-33187 M, 1:2000, Mouse; No. bs-0189R, 1:1000, Rabbit), Collagen I (Bioss, Item No. bs-7158R, 1:1000, Rabbit; No. AB260043 , 1:1000, Rabbit), Beclin-1 (Abcam, Item No. ab210498, 1:2000, Rabbit; No. ab62472, 1:1000, Rabbit), LC3B (CST, Item No. 3868s, 1:1000, Rabbit; No. 43566 S, 1:1000, Rabbit), and p62 (Bioss, Item No. bs-2951R, 1:1000, Rabbit) at 4°C with gentle shaking.

Techniques: In Vivo, In Vitro, Expressing, Control