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: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 1 Mahanine restores RASSF1A expression by demethylating its promoter and all three DNMTs control RASSF1A expression. A. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days. Methylation-specific PCR was performed to detect the methylated (M) and un-methylated (UM) status of RASSF1A promoter. B. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days, following which RASSF1A expression was assessed by RT-PCR. GAPDH was used as an internal control. C. PC3 cells were transfected with shRNA for DNMT1, DNMT3A, DNMT3B or scrambled shRNA. Forty-eight hours after transfection, cells were harvested for RT-PCR analyses to assess RASSF1A expression. GAPDH was used as an internal control. For DNMT3A, two shRNAs were used to confirm the result. D. BPH1 cells were transfected with expression vectors of DNMT1, DNMT3A, DNMT3B or empty vector control. Forty-eight hours after transfection cells were collected for RT-PCR analyses to determine RASSF1A, DNMT1, DNMT3A and DNMT3B expression levels. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Control, Methylation, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 2 Mahanine specifically down-regulates DNMT1 and DNMT3B. A. DNMT1, DNMT3A and DNMT3B cellular localization was visualized by immunofluorescent staining. PC3 cells were treated with DMSO (as control) or mahanine (10 μM) for 24 hours, following which they were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 488-tagged secondary antibodies and counterstained with propidium iodide. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or mahanine (10 μM) for 24 hours are shown (right panel). B. Cytoplasmic and nuclear fractions were separated from PC3 cells treated with DMSO or 10 μM mahanine for 24 hours. The isolated fractions were subjected to Western blot analysis to assess DNMT expression. The fold change in the expression of the respective DNMTs as compared to the control is indicated at the bottom of each immunoblot. Nucleolin and β-actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. C. PC3 and LNCaP cells were treated as indicated with DMSO or mahanine following which cells were lysed and the extracts were subjected to Western blot analysis to detect DNMT1, DNMT3B and DNMT3A protein levels (left). Quantitative estimations of the relative levels of DNMT1, DNMT3A and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin (right). Columns, mean; bars, SEM. *p < 0.05, significantly different from control. D. PC3 and LNCaP cells were treated with DMSO or 10 and 20 μM mahanine, respectively for 24 hours. Subsequently, cells were harvested for RT-PCR analysis to measure DNMT1 and DNMT3B expression. GAPDH was used as an internal control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Staining, Control, Incubation, Fluorescence, Microscopy, Isolation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 3 Mahanine degrades DNMTs via the ubiquitin-proteasomal pathway. A. PC3 cells were treated with 10 μM mahanine and 20 μM Z-VAD-FMK for 24 hours after which cellular protein lysates were subjected to Western blot analysis to detect DNMT1 and DNMT3B protein levels, β-actin was used as a loading control. B. Chymotrypsin-like proteasomal activity was measured in PC3 cells treated as indicated with mahanine and MG132 for 24 hours. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO control. C. LNCaP and PC3 cells were treated with the indicated doses of mahanine for 24 hours with or without MG132 (5 μM). Cell lysates were analyzed for DNMT1 and DNMT3B expression by Western blot. β-actin was used as a loading control. D. PC3 cells were treated with MG132 (5 μM) in the absence and presence of mahanine (10 μM) for 24 hours. Cell lysates were subjected to immunoprecipitation (IP) of DNMT1 or DNMT3B and immunoblotted (IB) for poly-ubiquitin, DNMT1 and DNMT3B.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Activity Assay, Expressing, Immunoprecipitation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 4 Mahanine down-regulates pAkt levels and PI3K/Akt inhibitor wortmannin reduces DNMT1 and DNMT3B protein levels. A. PC3 and LNCaP cells were treated with mahanine as indicated. The cell lysates were subjected to Western blot analysis for phospho Akt (pAkt), total Akt. Β-actin was used as a loading control. B. PC3 and LNCaP cells were treated with wortmannin (1 μM) for 24 hours. Cell lysates were subjected to Western blot analysis to measure DNMT1, DNMT3B, pAkt and total Akt levels. β-actin was used as a loading control. The fold change in expression of the respective proteins compared to control is indicated below. C. PC3 cells were treated with DMSO or wortmannin (1 μM) for 24 hours. DNMT1 and DNMT3B cellular localization was visualized by immunofluorescent staining. After 24h of treatment, cells were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 594-tagged secondary antibodies and counterstained with DAPI. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or wortmannin (1 μM) for 24 hours are shown (right panel).

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Western Blot, Control, Expressing, Staining, Incubation, Fluorescence, Microscopy

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 5 Mahanine disrupts the interaction of pAkt with DNMT1 and DNMT3B and constitutively active Akt (CA-Akt) stabilizes their cellular levels in the presence of mahanine. A. and B. LNCaP cells were treated with MG132 with or without 20 μM mahanine for 24 hours and the cell homogenates were subjected to co-immunoprecipitation (IP) for DNMT1 or DNMT3B and immunoblotted (IB) for phosphor-serine (pSer), pAkt, total Akt, DNMT1 and DNMT3B. C. BPH1 cells were transfected with an empty vector or a constitutively active Akt (CA-Akt) expression vector for 24 hours then were treated with or without mahanine (10 μM) for another 24 hours. Levels of total Akt, pAkt, DNMT1 and DNMT3B proteins were measured through Western blots. β-actin was used as a loading control. Quantitative estimations of relative levels of DNMT1 and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO treated control.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 6 Mahanine restores RASSF1A expression by degrading DNMTs via Akt. Prostate cancer cells express high levels of activated Akt, which phosphorylates and stabilizes DNMT1 and DNMT3B against proteasomal degradation. DNMTs enter the nucleus and methylate the promoter of RASSF1A gene to silence the expression of RASSF1A. Treatment of mahanine inhibits PDK1 and thereby prevents activation of Akt, which in turn compromises the stability of DNMTs, increases their ubiquitination and induces proteasomal degradation. In the absence of DNMT1 and DNMT3B, the RASSF1A promoter is demethylated and its expression is restored in prostate cancer cells. GF: Growth factor; RTK: Receptor tyrosine kinase; TFs: Transcription factors; P: Phosphorylated; M: Methylated; Ub: Ubiquitinated.

Article Snippet: After blocking (0.2% BSA) cells were incubated with the primary antibodies (DNMT1, DNM T3A or DNMT3B #Sc376043 from Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with the Alexa Fluor 488 or 594 conjugated secondary antibodies (Molecular probes/Invitrogen, Carlsbad, CA) for 1 hour.

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

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: Molecular Therapy. Nucleic Acids

Article Title: The roles of DNA methylation and hydroxymethylation at short interspersed nuclear elements in the hypothalamic arcuate nucleus during puberty

doi: 10.1016/j.omtn.2021.07.006

Figure Lengend Snippet: DNA methylation of SINEs in Htr6 promoters and Htr6 expression (A and C) DNA methylation of two CpG islands at the Htr6 promoter affected by DNMT RNA interference in wild-type GT1-7 cells (A) and GT1-7 SINE-CpG-KO1 cells (C), as observed through the bisulfite pyrosequencing assay. (B) DNA methylation of SINEs at the Htr6 promoter affected by DNMT RNA interference, as determined through the bisulfite pyrosequencing assay. (D and E) The levels of mRNA (D) and protein (E) in Htr6 induced by DNMT1 RNA interference in wild-type GT1-7 cells and GT1-7 SINE-CpG-KO1 cells. (F) DNA methylation of two CpG islands at the Htr6 promoter affected by L-tryptophan in wild-type GT1-7 cells and GT1-7 SINE-CpG-KO1 cells, as determined through the bisulfite pyrosequencing assay. (G and H) The levels of mRNA (G) and protein (H) in Htr6 induced by L-tryptophan in wild-type GT1-7 cells and GT1-7 SINE-CpG-KO1 cells. si, siRNA; WT, wild-type GT1-7 cells; KO, GT1-7 SINE-CpG-KO1 cells; Try, L-tryptophan; 1–25, 25 CpG sites distributed within two CpG islands in the Htr6 promoter as detected in this study. The qPCR data are presented as the mean ± SEM of three separate experiments. ∗p < 0.05 in Student’s t test.

Article Snippet: The blocked membranes were then incubated with primary antibodies against Dnmt1 (1:2,000, NB100-56519, Novus Biologicals, Centennial, CO, USA), Htr6 (1:1,500, NBP1-46557, Novus), Kiss1 (1:2,000, NBP1-45672, Novus), and Gapdh (1:5,000, 5174, Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, followed by incubation with the appropriate secondary antibodies (horseradish peroxidase-conjugated rabbit anti-mouse diluted at 1:10,000 and donkey anti-rabbit diluted 1:5,000, Cell Signaling Technology) for 30 min at RT.

Techniques: DNA Methylation Assay, Expressing, Pyrosequencing Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: The roles of DNA methylation and hydroxymethylation at short interspersed nuclear elements in the hypothalamic arcuate nucleus during puberty

doi: 10.1016/j.omtn.2021.07.006

Figure Lengend Snippet: Effect of NR5A2 on SINE methylation at the Htr6 promoter (A) DNA motif analysis of SINEs whose DNA methylation was positively correlated with transcription of adjacent genes using the JASPAR tool. (B) The occupancy of NR5A2 and DNMT1 on the Htr6 promoter in GT1-7 cells by ChIP-qPCR assay. ↑, ectopic overexpression; ↓, RNA interference. The qPCR data are presented as the mean ± SEM of three separate experiments. ∗p < 0.05 compared with L-tryptophan treatment. #p <0.05, representing significant differences between NR5A2 overexpression or blocking treatment.

Article Snippet: The blocked membranes were then incubated with primary antibodies against Dnmt1 (1:2,000, NB100-56519, Novus Biologicals, Centennial, CO, USA), Htr6 (1:1,500, NBP1-46557, Novus), Kiss1 (1:2,000, NBP1-45672, Novus), and Gapdh (1:5,000, 5174, Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, followed by incubation with the appropriate secondary antibodies (horseradish peroxidase-conjugated rabbit anti-mouse diluted at 1:10,000 and donkey anti-rabbit diluted 1:5,000, Cell Signaling Technology) for 30 min at RT.

Techniques: Methylation, DNA Methylation Assay, ChIP-qPCR, Over Expression, Blocking Assay

Journal: PLoS ONE

Article Title: miRNA-720 Controls Stem Cell Phenotype, Proliferation and Differentiation of Human Dental Pulp Cells

doi: 10.1371/journal.pone.0083545

Figure Lengend Snippet: List of primer pairs used for real time RT-PCR analysis.

Article Snippet: DPCs were seeded in 96-well plates, transfected with anti-miR-720, mimic-miR-720 or the respective negative controls, and cultured for 24 h. Immunocytochemical analyses were performed according to methods described previously , using primary antibodies against DNMT1 , , DNMT3A , DNMT3B (Imgenex Corporation, San Diego, CA, USA), NANOG (ReproCELL, Boston, MA, USA) or isotype control IgG.

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: miRNA-720 Controls Stem Cell Phenotype, Proliferation and Differentiation of Human Dental Pulp Cells

doi: 10.1371/journal.pone.0083545

Figure Lengend Snippet: A) Quantitative assessment of miR-720 levels. *** P<0.001, One way ANOVA/Tukey. Results are representative of at least 3 independent experiments. B) mimic-miR-720 reduced mRNA levels of NANOG , but increased the levels of DNMT3B and DNMT1 mRNA. No significant changes were observed in DNMT3A mRNA upon miR-720 transfection. * P<0.05, ** P<0.01, NS = non-significant, unpaired t -test, compared to si-control group. Results are representative of at least 3 independent experiments. C) Representative images of immunocytochemistry for NANOG protein levels. D) Quantitative analysis of NANOG or DNMTs positive cells. Mimic-miR-720 significantly reduced the number of NANOG positive cells, while increasing those for DNMT3B. There was no significant change in the percentages of DNMT1 or DNMT3A positive cells. Results in C–D are representative of at least 3 independent experiments. Quantitative analysis was performed on 500 cells/well, with at least triplicate samples. * P<0.05, ** P<0.01, NS = non-significant, unpaired t -test, compared to si-control group. G) Transfection of mimic-miR-720 decreased the percentage of SSEA-4 + cells. Results are representative of 3 independent experiments.

Article Snippet: DPCs were seeded in 96-well plates, transfected with anti-miR-720, mimic-miR-720 or the respective negative controls, and cultured for 24 h. Immunocytochemical analyses were performed according to methods described previously , using primary antibodies against DNMT1 , , DNMT3A , DNMT3B (Imgenex Corporation, San Diego, CA, USA), NANOG (ReproCELL, Boston, MA, USA) or isotype control IgG.

Techniques: Transfection, Control, Immunocytochemistry

Journal: PLoS ONE

Article Title: miRNA-720 Controls Stem Cell Phenotype, Proliferation and Differentiation of Human Dental Pulp Cells

doi: 10.1371/journal.pone.0083545

Figure Lengend Snippet: A) Quantitative assessment of the knockdown efficiency of miR-720. *** P<0.001, One way ANOVA/Tukey. Results are representative of at least 3 independent experiments. B) Knockdown of miR-720 increased the levels of NANOG transcripts, but reduced significantly the levels of DNMT3A , DNMT3B and DNMT1 transcripts. * P<0.05, ** P<0.01, *** P<0.001, unpaired t -test, compared to si-control group. Results are representative of at least 3 independent experiments. C) Knockdown of miR-720 increased the percentage of cells positive for SSEA-4. Results are representative of 3 independent experiments. D) Quantitative immunocytochemical analysis of NANOG, and DNMTs positive cells upon knockdown of miR-720. Anti-miR-720 increased the percentage of NANOG positive cells, but induced no significant changes in the percentage of DNMTs positive cells. NS = non-significant, unpaired t -test, compared to si-control group. Results are representative of at least 3 independent experiments. Quantitative analysis was performed on 500 cells/well, at least with triplicate samples.

Article Snippet: DPCs were seeded in 96-well plates, transfected with anti-miR-720, mimic-miR-720 or the respective negative controls, and cultured for 24 h. Immunocytochemical analyses were performed according to methods described previously , using primary antibodies against DNMT1 , , DNMT3A , DNMT3B (Imgenex Corporation, San Diego, CA, USA), NANOG (ReproCELL, Boston, MA, USA) or isotype control IgG.

Techniques: Knockdown, Control

Journal: NAR Cancer

Article Title: GSK-3484862, a DNMT1 degrader, promotes DNMT3B expression in lung cancer cells

doi: 10.1093/narcan/zcaf018

Figure Lengend Snippet: GSK-3484862 (labeled as GSK862) depletes DNMT1 protein and increases DNMT3B protein. ( A ) Mass spectrometry analysis of total 5mC content in A549 cells expressed as a percentage (i.e. number of 5mC-modified residues divided by the total number of cytosine residues × 100). The DMSO controls at different time points were averaged. ( B ) GSK862 induced hypomethylation, as determined by pyrosequencing, at three genomic loci ( CDX1 , NKX2-6 , and RASSF1A ) and at multiple transposable LINE-1 elements in A549 cells, as a function of treatment time and variation of compound concentrations. ( C ) Western blots showing endogenous levels of DNMT1 and DNMT3B in A549 cells following a 2-day treatment with GSK862 concentrations ranging from 0.02 to 2.0 μM. Note that the sample amount in the first lane was half of that in the other lanes. ( D ) Western blots showing increases of DNMT3B isoforms (indicated by arrows for long and short) and no changes in DNMT3A after treatment with GSK862. ( E ) Plots of intensities of protein bands of DNMT3A and DNMT3B shown in panel (D). ( F ) Concentration-dependent effect of GSK-3685032 (labeled as GSK032) in A549 cells. ( G ) Colony formation assay of A549 cells treated at the indicated doses of GSK862 for 10 days.

Article Snippet: The following primary antibodies were used in this study: DNMT1 [Cell Signaling Technology (CST), Cat. #5032], DNMT3A (CST, Cat. #3598), DNMT3B (CST, Cat. #72335), H3 (CST, Cat. #14269), GAPDH (CST, Cat. #2118), actin (Sigma, Cat. #A2228), and vinculin (Sigma, Cat. #SAB4200729).

Techniques: Labeling, Mass Spectrometry, Modification, Western Blot, Concentration Assay, Colony Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 4. Dnmt1 deficiency promotes oxidative metabolism across hepatocyte zonation. A) UMAP visualization of hepatocyte zonation representing the periportal zone, midzonal zone (midzone), and pericentral zone. Nonhepatocytes: NHCs. B) Dot plot of differentially expressed zonation marker genes across the three zones. C) The proportion of each hepatocyte subpopulation in the total liver cells of LD1KO mice and fl/flmice. D) Heatmap of gene expression of oxidative phosphorylation across the three hepatocyte zones in LD1KO mice and fl/flmice (left panel) and heatmap of gene expression of fatty acid oxidation across the three hepatocyte zones in LD1KO mice and fl/flmice (right panel).

Article Snippet: The cleared cell lysates containing 2 mg total protein was incubated with the DNMT1 primary antibody (IMG-261A, IMGENEX) and protein A/G PLUS-Agarose at 4°C overnight, washed with PBS, boiled and loaded in SDS-PAGE and immunoblotted with the ubiquitin antibody (sc-8017, Santa Cruz).

Techniques: Marker, Gene Expression, Phospho-proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 5. DNA methylation regulates the expression of fatty acid oxidative genes. A) Quantitative RT-PCR analysis of fatty acid oxidative gene expression in the liver of LD1KO and fl/flmice. B) Quantitative RT-PCR analysis of fatty acid oxidative gene expression in the liver of LD3aKO and fl/flmice. C) Quantitative analysis of fatty acid oxidative gene expression in the liver of LT2KO and fl/flmice. D) Oxygen consumption rate (OCR) of primary hepatocytes isolated from LD1KO and fl/flmice. Dnmt1 fl/fl, Dnmt3a fl/fl, or Tet2 fl/flmice were intravenously injected with AAV-TBG-Cre virus to generate LD1KO, LD3aKO or LT2KO mice respectively, which were then fed with HFD as described in Figure 2. All data are expressed as mean±SEM. n = 5-8; *p < 0.05 versus fl/fl.

Article Snippet: The cleared cell lysates containing 2 mg total protein was incubated with the DNMT1 primary antibody (IMG-261A, IMGENEX) and protein A/G PLUS-Agarose at 4°C overnight, washed with PBS, boiled and loaded in SDS-PAGE and immunoblotted with the ubiquitin antibody (sc-8017, Santa Cruz).

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Gene Expression, Isolation, Injection, Virus

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 6. Methylation of the Klb promoter is enhanced by HFD. A) RRBS analysis reveals an enhanced DNA methylation rate at the Klb promoter by HFD as shown in UCSC Genome Browser on Mouse (NCBI37/mm9) Assembly. B) Pyrosequencing analysis of the DNA methylation at the CpG sites at the Klb promoter in the liver of HFD- and LFD-fed mice (n = 8). C) Quantitative RT-PCR analysis of Klb mRNA expression in liver of HFD- and LFD-fed mice (n = 6–8). D) The association of the peaks of the chromatin accessibility at the Klb promoter was analyzed by ATAC-seq and the reads of the Klb mRNA expression analyzed by RNA-seq in the liver of HFD- and LFD-fed mice. () Luciferase activity of the Klb promoter regulated by DNA methylation (n = 8). F) DNMT1 binding to the Klb promoter was measured by ChIP assays (n = 4). G) DNMT3A binding to the Klb promoter measured by ChIP assays (n = 4). 6-week-old male C57BL/6J mice were fed either LFD or HFD for 12 weeks. All data are expressed as mean±SEM. *p < 0.05 versus LFD. H) Heatmap of Klb and Dnmt1 expression across the three hepatocyte zones (periportal zone, midzone, and pericentral zone) in LD1KO mice and fl/fl mice with snRNA-seq analysis. I) Quantitative RT-PCR analysis of Klb mRNA expression in Dnmt1-deficient liver (left), Dnmt3a-deficient liver (middle), or Tet2-deficient liver (right) (n = 5-9). J) Pyrosequencing analysis of DNA methylation rate in the CpG sites of the Klb promoter in the liver of LD1KO mice and fl/flmice (n = 8). K) Pyrosequencing analysis of DNA methylation rate in the CpG sites of the Klb promoter in the liver of LT2KO mice and fl/fl mice (n = 4-8). All data are expressed as mean ± SEM. *p < 0.05 versus fl/fl.

Article Snippet: The cleared cell lysates containing 2 mg total protein was incubated with the DNMT1 primary antibody (IMG-261A, IMGENEX) and protein A/G PLUS-Agarose at 4°C overnight, washed with PBS, boiled and loaded in SDS-PAGE and immunoblotted with the ubiquitin antibody (sc-8017, Santa Cruz).

Techniques: Methylation, DNA Methylation Assay, Quantitative RT-PCR, Expressing, RNA Sequencing, Luciferase, Activity Assay, Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 7. Dnmt1 or Dnmt3a deficiency increases FGF15 or FGF21-stimulated fatty acid oxidative gene expression in primary hepatocytes. A) Quantitative RT-PCR analysis of fatty acid oxidative gene expression in the Dnmt1 deficient hepatocytes (n = 3-6). B) Quantitative RT-PCR analysis of fatty acid oxidative gene expression in the Dnmt3a deficient hepatocytes (n = 5-6). Primary hepatocytes were isolated from Dnmt1- or Dnmt3a-floxed mice and were infected with AAV8-TBG-Cre AAV or control virus to generate Dnmt1 or Dnmt3a-deficient hepatocytes. The hepatocytes were then treated with FGF15 or FGF21 for 4 h. All data are expressed as mean±SEM. *p < 0.05 versus fl/fl.

Article Snippet: The cleared cell lysates containing 2 mg total protein was incubated with the DNMT1 primary antibody (IMG-261A, IMGENEX) and protein A/G PLUS-Agarose at 4°C overnight, washed with PBS, boiled and loaded in SDS-PAGE and immunoblotted with the ubiquitin antibody (sc-8017, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Isolation, Infection, Control, Virus

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 9. Regulation of DNMT1 protein stability by UBE3A. A) The association of the peaks of the chromatin accessibility at the Ube3a promoter analyzed by ATAC-seq and the reads of the Ube3a mRNA expression analyzed by RNA-seq in the liver of HFD-fed or LFD-fed mice. B) Immunoblotting analysis of UBE3A in the liver of mice fed LFD or HFD. All data are expressed as mean ± SEM. n = 5; *p < 0.05 versus LFD. C) Immunoblotting analysis of DNMT1 and DNMT3A protein in the liver of mice infected with AAV Ube3a expression virus. All data are expressed as mean ± SEM. n = 5; *p < 0.05 versus control. D) UBE3A regulation of DNMT1 ubiquitination. Ube3a was knocked down by shRNA or re-expressed into the knockdown cells with Ube3a expression vectors or overexpressed with Ube3a expression vectors in HEK293 cells. DNMT1 protein was immunoprecipitated with an anti-DNMT1 antibody and followed by immunoblotting with an anti-ubiquitin antibody. E) UBE3A promotes DNMT1 protein degradation in a cycloheximide (CHX) chase assay. The CHX chase assay was conducted in the HEK293 cells with Ube3a knockdown or overexpression. The cells were pre-treated with CHX and then harvested in a time course ranging from 0 to 16 h.

Article Snippet: The cleared cell lysates containing 2 mg total protein was incubated with the DNMT1 primary antibody (IMG-261A, IMGENEX) and protein A/G PLUS-Agarose at 4°C overnight, washed with PBS, boiled and loaded in SDS-PAGE and immunoblotted with the ubiquitin antibody (sc-8017, Santa Cruz).

Techniques: Expressing, RNA Sequencing, Western Blot, Infection, Virus, Control, Ubiquitin Proteomics, shRNA, Knockdown, Immunoprecipitation, Over Expression