Journal: Molecular medicine reports

Article Title: Procaine stimulates aquaporin‑5 expression in human salivary gland ductal cells via the suppression of DNA methyltransferase‑1.

doi: 10.3892/mmr.2018.8821

Figure Lengend Snippet: Figure 5. Expression and activation levels of DNMTs in procaine‑treated NS‑SV‑DC cells. (A) Western blot analysis of DNMT1, DNMT3A and DNMT3B proteins in procaine (2 µM)‑treated NS‑SV‑DC cells. (B) Methylation activity assay of procaine‑treated NS‑SV‑DC cells. The methylation of DNMT1 procaine‑treated NS‑SV‑AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity assay kit. The results are expressed as OD at a wavelength of 450 nm and are the mean ± standard deviation of three separate experiments. *P<0.05, **P<0.01, ***P<0.001 vs 0 h within procaine‑treated (500 or 2 µM) group. DNMT, DNA methyltransferase; NS‑SV‑DC, normal human salivary gland ductal cells; OD, optical density.

Article Snippet: The protein expression of DNMTs was analyzed by the same method with mouse monoclonal IgG1 DNMT1 (1:100), DNMT3A (dilution: 1:100; cat. no. sc‐373905, Santa Cruz Biotechnology, Inc.) and DNMT3B (dilution: 1:100; cat. no. sc‐70984, Santa Cruz Biotechnology, Inc.) antibodies and corresponding HRP-conjugated antibodies (dilution: 1:1,000; cat. no. sc‐516102). β-actin protein was used as internal reference and detected as aforementioned with mouse monoclonal β‐actin antibody (dilution: 1:100; cat. no. sc‐8432, Santa Cruz Biotechnology, Inc.) and corresponding HRP‐conjugated antibodies (dilution: 1:1,000; cat. no. sc‐516102).

Techniques: Expressing, Activation Assay, Western Blot, Methylation, Activity Assay, Standard Deviation

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