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: Journal of Cellular and Molecular Medicine

Article Title: TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

doi: 10.1111/j.1582-4934.2011.01456.x

Figure Lengend Snippet: Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U -test. * P < 0.05 compared with control cells.

Article Snippet: The membranes were blocked with 2% skim milk and incubated with goat anti-human AQP5 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human Dnmt1 antibody (Santa Cruz Biotechnology), rabbit anti-human Dnmt3a antibody (Santa Cruz Biotechnology), rabbit anti-human Dnmt3b antibody (Santa Cruz Biotechnology) or rabbit anti-human IκBα antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Methylation, Activity Assay, MANN-WHITNEY, Control

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: DNMT1 primers.

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: Sequencing

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: Transduction

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: shRNA containing vectors used to knockdown DNMT1 expression Dnmt1 protein expression (derived from GenBank Accession: {"type":"entrez-nucleotide","attrs":{"text":"NM_001379","term_id":"1677703423","term_text":"NM_001379"}} NM_001379 ).

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: shRNA, Expressing, Derivative Assay, Sequencing, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Effect of high glucose on DNA methylation of Mlh1 promoter and its transcriptional regulation. HRECs, incubated in high glucose, were analyzed for (a) 5mC levels at Mlh1 promoter by methylated DNA immunoprecipitation method, (b, c) Dnmt1 and Sp1 binding by ChIP technique, and (d) Mlh1 gene transcripts by qPCR. Each measurement was made in duplicate in three to four samples per group. The values obtained from cells in NG are considered as 1. NG and HG = 5 mM and 20 mM glucose, respectively; D-si and SC = cells transfected with Dnmt1-siRNA and control negative RNA, respectively, and incubated in HG; Aza = 1 μM Aza-2′-deoxyxytidine. *P < 0.05 versus NG and #P < 0.05 versus HG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Incubation, Methylation, Immunoprecipitation, Binding Assay, Transfection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Regulation of DNA methylation and base mismatches. DNA from HRECs was amplified using semi-qPCR for the D-loop and digested with mismatch-specific surveyor endonuclease. The samples were analyzed on a 2% agarose gel. The parent amplicon band intensity was quantified by densitometry, and the values obtained from cells in NG was considered as 100%. Data are represented as mean ± SD, and each measurement was made in duplicate in three to five samples in each group. Mismatches and the parent amplicon band intensity from (a) Dnmt1-siRNA transfected HRECs and (b) Aza-treated HRECs. NG and HG = HRECs in 5 mM and 20 mM glucose, respectively; D-si and SC = cells transfected with Dnmt1-siRNA and negative control RNA, respectively; Aza = 1 μM 5-Aza-2′-deoxyxytidine. *P < 0.05 compared with NG; #P < 0.05 compared with HG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Amplification, Agarose Gel Electrophoresis, Transfection, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Differential DNA methylation of the Mlh1 promoter in HRECs incubated in high glucose. Four CpG-rich regions of the proximal promoter of Mlh1 (−659 to 72; R1-R4) were analyzed for (a) 5mC levels by methylated DNA immunoprecipitation method and (b) Dnmt1 binding by ChIP using IgG (represented as ^) as an antibody control. The values are represented as fold change, and the values obtained from the cells in NG are considered as one. NG and HG = 5 mM and 20 mM glucose, respectively; Aza = 1μM Aza-2′-deoxycytidine. *P < 0.05 compared to NG.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Incubation, Methylation, Immunoprecipitation, Binding Assay, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: DNA methylation of the Mlh1 promoter in diabetes and its regulation by Dnmt inhibitors. Retinal microvessels from diabetic mice receiving intravitreal administration of Dnmt1-siRNA (D-si) or intraperitoneal injection of Aza-2′-deoxyxytidine (Aza) were analyzed for (a) Mlh1 mRNA levels by qPCR and (b) 5mC levels at the promoter region of Mlh1 by a methylated DNA immunoprecipitation kit. Values are calculated as fold change as compared to normal and are represented as mean ± SD. Nor and Diab = nondiabetic control and streptozotocin-induced diabetic mice, respectively. *P < 0.05 compared to normal and #P < 0.05 compared to diabetes.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: DNA Methylation Assay, Injection, Methylation, Immunoprecipitation, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

doi: 10.1167/iovs.19-27602

Figure Lengend Snippet: Methylation of MLH1 in human donors with diabetic retinopathy. Retinal microvessels from human donors with documented diabetic retinopathy (DR) and age-matched nondiabetic donors (Norm) were analyzed for MLH1 and DNMT1 gene transcripts by qPCR using β-actin as a housekeeping gene (a). The MLH1 promoter was analyzed for (b) 5mC levels using a methylated DNA immunoprecipitation method and (c) Dnmt1 binding by ChIP technique. Values are mean ± SD of at least six donors in each group, with each measurement made in duplicate, and are presented as fold change compared to nondiabetic donors. ^ IgG antibody control. *P < 0.05 compared to nondiabetic control.

Article Snippet: L-glucose (20 mM), instead of 20 mM D-glucose, was used as the osmotic/metabolic control., , A group of cells from the fifth to sixth passage were transfected with Dnmt1- siRNA (cat. no. SC-35204, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (cat. no. SC-29528, Santa Cruz Biotechnology).

Techniques: Methylation, Immunoprecipitation, Binding Assay, Control

Journal: Cell Genomics

Article Title: Genome-wide classification of epigenetic activity reveals regions of enriched heritability in immune-related traits

doi: 10.1016/j.xgen.2023.100469

Figure Lengend Snippet:

Article Snippet: Following blotting onto nitrocellulose membrane, anti-actin (clone C4, Millipore) and anti-DNMT1 (clone JF09-89, Novus Biologicals) were used for protein detection, with staining with IRDye 680LT- and 800CW-conjugated secondary antibodies and visualization using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Recombinant, Sequencing, Positive Control, Software