Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: NAR Cancer

Article Title: GSK-3484862 targets DNMT1 for degradation in cells

doi: 10.1093/narcan/zcad022

Figure Lengend Snippet: GSK( 1 ) targets DNMT1 for degradation in A549 cells. ( A ) Chemical structures of GSK( 1 ) and GSK( 2 ) and DNMT1-bound GSK( 2 ) with the inhibitor shown in yellow and the DNMT1 active-site loop shown in magenta (initially reported in ( , )). ( B ) Western blots showing endogenous levels of DNMT1, DNMT3A and DNMT3B following treatment with 2 and 4 μM GSK( 1 ) for 24 h. While DNMT3A appeared to be slightly reduced, subsequent experiments revealed no significant changes in DNMT3A level ( and Figure ). ( C ) Relative gene expression of DNMT1 in the presence and absence of GSK( 1 ) were analyzed by RT-qPCR and normalized relative to IPO8 ( N = 3). The corresponding western blot is shown in lower panel for three repeats of DMSO (in blue dots) and GSK( 1 )-treated samples (in red dots). ( D ) Cell growth as determined by cell viability assay over 2 days. ( E ) Time-dependent depletion of DNMT1 in cells treated with GSK( 1 ). ( F ) Comparison of the potency of GSK( 1 ) and GSK( 2 ) in inducing DNMT1 depletion. ( G ) GSK( 2 ) showed an enhanced cell toxicity compared to GSK( 1 ) at 4 μM in A549 cells. ( H ) Concentration-dependent effect of GSK( 1 ). ( I ) Time-dependent effect of GSK( 1 ) at 80 nM. ( J ) Little effect of GSK( 1 ) at low concentrations (3.2 and 16 nM). In panels H–J, the two DMSO lanes as indicated by a bracket are the 2× dilution (1/2) of cell lysates.

Article Snippet: The primary antibodies used in this study were: DNMT1 [(Cell Signaling Technology (CST), #5032), DNMT3A (CST, #3598), DNMT3B (CST, #67259), H3 (CST, #14269), GAPDH (CST, #2118), Oct4 (CST, #4286), PCNA (CST, #13110), Uhrf1 (CST, #12387), Actin (Sigma-Aldrich, A2228), Myc tag (Sigma-Aldrich, M4439), Flag tag (Sigma-Aldrich, F3165) and 5- methylcytosine (5mC) (Active Motif, #39649).

Techniques: Western Blot, Gene Expression, Quantitative RT-PCR, Viability Assay, Comparison, Concentration Assay

Journal: NAR Cancer

Article Title: GSK-3484862 targets DNMT1 for degradation in cells

doi: 10.1093/narcan/zcad022

Figure Lengend Snippet: GSK( 1 ) induces DNA hypomethylation and proteasome-dependent and DNA synthesis-dependent loss of DNMT1 protein. ( A ) Dot blot assay using an anti-5mC antibody to detect 5mC in genomic DNA. The lower panel shows the same membrane used in top panel stained with methylene blue to verify DNA loading. ( B ) GSK( 1 )-induced hypomethylation at three genomic loci by pyrosequencing. Three technical replicates were measured for each sample. The three DMSO controls at 12, 24 and 48 h were averaged. ( C ) Mass spectrometry analysis of total 5mC content expressed as a percentage (i.e. number of 5mC-modified residues divided by the total number of cytosine residues × 100). ( D ) A549 cells were treated with GSK( 1 ) (left), MG132 (middle) or both (right) up to 24 h and subjected to immunoblotting. ( E , F ) For treatment of 12 h, the effects of GSK( 1 ) or DAC on their own (panel E) as compared with cells that had been treated with the DNA synthesis inhibitor aphidicolin for 24 h prior to treatment with GSK( 1 ) or DAC (panel F). ( G ) As in panel E and F, the effect of GSK( 1 ) or DAC treatment of 24 h, in the absence (left) and presence of aphidicolin (right). In panels F and G, the two DMSO lanes as indicated by a bracket are the 2× dilution (1/2) of cell lysates.

Article Snippet: The primary antibodies used in this study were: DNMT1 [(Cell Signaling Technology (CST), #5032), DNMT3A (CST, #3598), DNMT3B (CST, #67259), H3 (CST, #14269), GAPDH (CST, #2118), Oct4 (CST, #4286), PCNA (CST, #13110), Uhrf1 (CST, #12387), Actin (Sigma-Aldrich, A2228), Myc tag (Sigma-Aldrich, M4439), Flag tag (Sigma-Aldrich, F3165) and 5- methylcytosine (5mC) (Active Motif, #39649).

Techniques: DNA Synthesis, Dot Blot, Membrane, Staining, Mass Spectrometry, Modification, Western Blot

Journal: NAR Cancer

Article Title: GSK-3484862 targets DNMT1 for degradation in cells

doi: 10.1093/narcan/zcad022

Figure Lengend Snippet: Effect of the GSK compounds in a range of cancer cells. ( A ) Western blot confirms decreased DNMT1 levels in four cell lines derived from solid tumors. ( B ) Cell viability (relative to DMSO treatment) after the myeloid leukemia cell lines MOLM13 (top panel) and THP1 (bottom panel) were treated with GSK( 1 ) with concentrations starting from 50 μM (with 2x dilution until 48 nM) for three days ( N = 3 independent experiments with quadruplicate; mean ± SD). (C) As in panel B but with GSK( 2 ) with concentrations starting from 25 μM with 2x dilution until 24 nM. ( D ) Western blot showing decreased levels of DNMT1 in MOLM13 and THP1, treated with GSK( 1 ) or GSK( 2 ) at two different concentrations with duplications. ( E–G ) Treatment with GSK( 1 ) at 4 μM for 1 day in the indicated myeloid leukemia cell lines showing cell viability ( E ), relative DNMT1 mRNA ( F ) and DNMT1 protein levels ( G ). DMSO samples are in blue dots and GSK(1)-treated samples are in red dots.

Article Snippet: The primary antibodies used in this study were: DNMT1 [(Cell Signaling Technology (CST), #5032), DNMT3A (CST, #3598), DNMT3B (CST, #67259), H3 (CST, #14269), GAPDH (CST, #2118), Oct4 (CST, #4286), PCNA (CST, #13110), Uhrf1 (CST, #12387), Actin (Sigma-Aldrich, A2228), Myc tag (Sigma-Aldrich, M4439), Flag tag (Sigma-Aldrich, F3165) and 5- methylcytosine (5mC) (Active Motif, #39649).

Techniques: Western Blot, Derivative Assay

Journal: NAR Cancer

Article Title: GSK-3484862 targets DNMT1 for degradation in cells

doi: 10.1093/narcan/zcad022

Figure Lengend Snippet: Effect of GSK( 1 ) in mESCs. ( A ) Western blot showing GSK( 1 )-induced Dnmt1 depletion. ( B ) Southern blot after digestion of genomic DNA from cells in panel A with the methylation-sensitive restriction enzyme HpaII, which shows GSK( 1 )-induced hypomethylation of the minor satellite repeats. ( C ) RT-qPCR data showing no change in Dnmt1 mRNA after treatment with GSK( 1 ). DMSO samples are in blue dots and GSK(1)-treated samples are in red dots. ( D ) Cell viability (relative to DMSO treatment) after WT and Dnmt1 −/− mESCs were treated with GSK( 1 ) (top panel) or GSK( 2 ) (bottom panel) with concentrations ranging from 20 nM to 12.5 μM (1:5 serial dilutions starting at 12.5 μM) for three days ( N = 3 independent experiments with triplicate; mean ± SD). ( E ) mESCs were treated with 2 μM of GSK( 1 ) for 24 h and then cultured in the absence of the compound for 4 additional days. Western blot shows complete recovery of DNMT1 protein by 1 d after removal of the compound. ( F ) Southern blotting analysis of the minor satellite repeats in cells from panel E showing gradual recovery of DNA methylation after removal of the compound. The two blots were from the same membrane with different exposure times. ( G ) Mouse Dnmt1, with the conserved catalytic motifs (I-X) and the location of the PC:SF mutation being shown. ( H ) Western blotting analysis of Dnmt1 −/− mESCs reconstituted with Myc-tagged Dnmt1 or the PC:SF mutant showing degradation of both WT and mutant Dnmt1 after GSK( 1 ) treatment (2 μM for 24 h). Two stable clones for each construct were tested. ( I ) Southern blotting analysis of the minor satellite repeats in cells from panel H showing restoration of DNA methylation by WT Dnmt1 (lanes 4 and 5), but not by the mutant (lanes 8 and 9), and the effect of GSK( 1 ) on DNA methylation.

Article Snippet: The primary antibodies used in this study were: DNMT1 [(Cell Signaling Technology (CST), #5032), DNMT3A (CST, #3598), DNMT3B (CST, #67259), H3 (CST, #14269), GAPDH (CST, #2118), Oct4 (CST, #4286), PCNA (CST, #13110), Uhrf1 (CST, #12387), Actin (Sigma-Aldrich, A2228), Myc tag (Sigma-Aldrich, M4439), Flag tag (Sigma-Aldrich, F3165) and 5- methylcytosine (5mC) (Active Motif, #39649).

Techniques: Western Blot, Southern Blot, Methylation, Quantitative RT-PCR, Cell Culture, DNA Methylation Assay, Membrane, Mutagenesis, Clone Assay, Construct

Journal: NAR Cancer

Article Title: GSK-3484862 targets DNMT1 for degradation in cells

doi: 10.1093/narcan/zcad022

Figure Lengend Snippet: Uhrf1 is required for GSK( 1 )-induced Dnmt1 degradation in mESCs. ( A ) Western blot showing that Dnmt1 is degraded in WT, but not Uhrf1 −/− , mESCs after GSK( 1 ) treatment (2 μM for 48 h). ( B ) Southern blotting analysis of the minor satellite repeats in cells from panel A showing that GSK( 1 ) induced hypomethylation in WT mESCs but did not induce further loss of methylation in Uhrf1 −/− mESCs. ( C ) Mouse Uhrf1, with the functional domains – ubiquitin-like (UBL) domain, tandem Tudor domain (TTD), plant homeodomain (PHD), SET- and RING-associated (SRA) domain, and Really Interesting New Gene (RING) domain – and the location of the H730A mutation being shown. ( D ) Western blotting analysis of Uhrf1 −/- mESCs reconstituted with Flag-tagged Uhrf1 or the H730A mutant showing that GSK( 1 ) treatment (2 μM for 24 h) resulted in partial Dnmt1 degradation in the Flag-Uhrf1 stable clone, but not in the Flag-H730A stable clone. ( E ) Western blot showing that Uhrf1 deficiency also prevented Dnmt1 depletion induced by decitabine treatment (1–5 μM for 48 h).

Article Snippet: The primary antibodies used in this study were: DNMT1 [(Cell Signaling Technology (CST), #5032), DNMT3A (CST, #3598), DNMT3B (CST, #67259), H3 (CST, #14269), GAPDH (CST, #2118), Oct4 (CST, #4286), PCNA (CST, #13110), Uhrf1 (CST, #12387), Actin (Sigma-Aldrich, A2228), Myc tag (Sigma-Aldrich, M4439), Flag tag (Sigma-Aldrich, F3165) and 5- methylcytosine (5mC) (Active Motif, #39649).

Techniques: Western Blot, Southern Blot, Methylation, Functional Assay, Ubiquitin Proteomics, Mutagenesis, Stable Transfection

Journal: EBioMedicine

Article Title: UVB induces cutaneous squamous cell carcinoma progression by de novo ID4 methylation via methylation regulating enzymes

doi: 10.1016/j.ebiom.2020.102835

Figure Lengend Snippet: Sequences of primers used in qPCR.

Article Snippet: The slides were first blocked with normal goat serum at room temperature for 30 min to minimize nonspecific staining, then incubated overnight with primary antibodies against DNMT1 (1:100, Absin Bioscience Inc., Shanghai, China), DNMT3A (1:50, CST, USA), DNMT3B (1:50, Abcam, UK), TET1 (1:100, Absin Bioscience Inc., Shanghai, China), TET2 (1:100, Absin Bioscience Inc., Shanghai, China), and TET3 (1:100, Abcam, UK).

Techniques: Sequencing

Journal: EBioMedicine

Article Title: UVB induces cutaneous squamous cell carcinoma progression by de novo ID4 methylation via methylation regulating enzymes

doi: 10.1016/j.ebiom.2020.102835

Figure Lengend Snippet: Altered expression of DNMTs and TETs in CSCCs compared with adjacent normal tissues. (a) The relative expression levels of DNMTs and TETs in CSCC and adjacent normal tissues. Data are shown as means ± SD. The statistical significance was assessed by the Student's t -test. (b) The Pearson's correlation between expression level (2 −△Ct ) or methylation level of ID4 and expression levels (2 −△△Ct ) of DNMT1, DNMT3A, DNMT3B, TET1, TET2, and TET3, which were measured by MSP and qPCR in the same patient sets.

Article Snippet: The slides were first blocked with normal goat serum at room temperature for 30 min to minimize nonspecific staining, then incubated overnight with primary antibodies against DNMT1 (1:100, Absin Bioscience Inc., Shanghai, China), DNMT3A (1:50, CST, USA), DNMT3B (1:50, Abcam, UK), TET1 (1:100, Absin Bioscience Inc., Shanghai, China), TET2 (1:100, Absin Bioscience Inc., Shanghai, China), and TET3 (1:100, Abcam, UK).

Techniques: Expressing, Methylation

Journal: Genetics Research

Article Title: Functional Analysis of DNMT1 SNPs ( rs2228611 and rs2114724 ) Associated with Schizophrenia

doi: 10.1155/2021/6698979

Figure Lengend Snippet: Evaluation of DNMT1 . (a) Violin plots showing relative transcript levels in peripheral blood monocytes of individuals with the haplotypes indicated on the X -axis. (b) Immunofluorescence of peripheral blood monocytes with antibodies to HISTONE H3 (green) and DNMT1 proteins (red). (c) Quantification of the signals obtained for DNMT1 after normalization for the signals obtained with HISTONE H3 in peripheral blood monocytes of three individuals each for the genotypes indicated.

Article Snippet: Primary antibodies for DNMT1 (Thermo Fisher, 60B1220.1) and HISTONE H3 (Cell Signaling Technologies, 4499) diluted in 1% BSA were added to the dishes and were incubated overnight at 4°C.

Techniques: Immunofluorescence

Journal: Cancers

Article Title: Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation

doi: 10.3390/cancers12020447

Figure Lengend Snippet: ( A ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to the indicated concentrations of 2b or 4c . Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as loading control. Blots are representative of two independent experiments. Right: Densitometric analysis of protein levels is reported. ( B ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to 4c at 1 µM and co-treated with bortezomib (when indicated) used at 10 nM. Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as a loading control. Blots are representative of three independent experiments. Right: Densitometric analysis of protein levels is reported. Data are represented as mean ± SEM. Significance is represented as * p < 0.05 related to the control.

Article Snippet: The following primary antibodies were used for immunoblotting: α-DNMT1 (Novus Biologicals, Littleton, CO, USA). α-DNMT3a (SantaCruz Bio-technologies, CA, USA) and α-GAPDH (Millipore Corp., Bedford, MA USA), used as a loading control.

Techniques: Western Blot, Expressing, Control

Journal: Journal of Cancer

Article Title: Hypermethylation of mitochondrial DNA facilitates bone metastasis of renal cell carcinoma

doi: 10.7150/jca.62278

Figure Lengend Snippet: mtDNA copy number and mitochondria DNMT1 expressions in BO and P RCC tumor specimens and cells. A. Relative mtDNA copy number in 15 pairs of BO and P specimens. B. Comparison of the average relative mtDNA copy number between the 15 cases. * P<0.05. C. Representative blots of DNMT1, DNMT3A, and DNMT3B in nuclear (Nucl) and mitochondrial (Mito) fractions of ACHN and OS-CR-2 cells. D. Semi-quantification of DNMT1 and DNMT3A in C. Expression was normalized to the respective internal reference markers (VDAC1 or Histone 3), protein expression in P cells was set as 1. * P<0.05.

Article Snippet: Primary antibodies against DNMT1 (Santa Cruz Biotech), DNMT3A (Abcam), DNMT3B (Abcam), GAPDH (Santa Cruz Biotech), VDAC1 (Abcam), and Histone 3 (Abcam) were used, donkey anti-rabbit/-mouse/-goat IgG (H&L) was used as second antibodies (Santa Cruz Biotech).

Techniques: Comparison, Expressing