Journal: Frontiers in Immunology

Article Title: Promotion of Treg/Th17 balance in MRL/lpr mice by Jianpi-Zishen Formula via modulation of DNMT1-mediated Foxp3 methylation

doi: 10.3389/fimmu.2025.1631631

Figure Lengend Snippet: DNA methylation levels of Foxp3 in the CD4 + T cells of MRL/lpr mice and the reducing effect of JPZS. (A) CPG islands of Foxp3 - TSDR in CD4 + T cells of MRL/lpr mice. (B) The effects of JPZS and 5-aza-CdR on the Foxp3 methylation levels in CD4 + T cells. (C) The effect of JPZS and 5-aza-CdR on the methylation levels of 10 CPG islands in the CD4 + T cells Foxp3 . (D) The transcription level of Foxp3 mRNA after 5-aza-CdR treatment. (E) Methyltransferase Dnmt1, Dnmt3a, and Dnmt3b activities were noted in the CD4 + T cells treated with 5-aza-CdR. (F) Methyltransferase Dnmt1, Dnmt3a, and Dnmt3b activities were observed in the CD4 + T cells treated with DC_517. (G) The expression level of the Dnmt1 protein after DC_517 treatment. (H) The transcription level of Foxp3 mRNA after DC_517 treatment. ****P<0.0001, ***P<0.001, *P<0.05.

Article Snippet: After transferring, the membranes were treated with a 10% skim milk solution in Tris-buffered saline with Tween and then exposed to primary antibodies targeting Dnmt1 (1:1000, Bioss Inc, bs-0678R) at 4°C for 12 hours.

Techniques: DNA Methylation Assay, Methylation, Expressing

Journal: Frontiers in Immunology

Article Title: Promotion of Treg/Th17 balance in MRL/lpr mice by Jianpi-Zishen Formula via modulation of DNMT1-mediated Foxp3 methylation

doi: 10.3389/fimmu.2025.1631631

Figure Lengend Snippet: DNMT1-mediated Foxp3 - TSDR methylation and reduction of JPZS. (A, B) The effect of JPZS on Dnmt1 activity and protein expression levels. (C) The effects of JPZS and DC_517 on Foxp3 methylation levels in CD4 + T cells. (D) The effects of JPZS and DC_517 on the methylation levels of 10 CpG islands in CD4 + T cells Foxp3 . (E) The effect of JPZS and DC_517 on the transcription level of Foxp3 mRNA in the peripheral blood CD4 + T cells of MRL/lpr mice. ****P<0.0001, ***P<0.001, **P<0.01.

Article Snippet: After transferring, the membranes were treated with a 10% skim milk solution in Tris-buffered saline with Tween and then exposed to primary antibodies targeting Dnmt1 (1:1000, Bioss Inc, bs-0678R) at 4°C for 12 hours.

Techniques: Methylation, Activity Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Promotion of Treg/Th17 balance in MRL/lpr mice by Jianpi-Zishen Formula via modulation of DNMT1-mediated Foxp3 methylation

doi: 10.3389/fimmu.2025.1631631

Figure Lengend Snippet: JPZS promotes Treg/Th17 balance by inhibiting DNMT1-mediated Foxp3 methylation. (A) The frequency of Th17 cells and Treg cells in splenocytes was denoted by flow cytometry analyses. (B, C) The effects of JPZS and DC_517 on the transcription levels of Foxp3 and RORγt mRNA in CD4 + T cells. (D) Effects of JPZS and DC_517 on serum cytokine levels in MRL/lpr mice. (G–I) The effects of JPZS and DC_517 on the levels of IgG, dsDNA, and C3 in the serum of MRL/lpr mice. ****P<0.0001, ***P<0.001, *P<0.05.

Article Snippet: After transferring, the membranes were treated with a 10% skim milk solution in Tris-buffered saline with Tween and then exposed to primary antibodies targeting Dnmt1 (1:1000, Bioss Inc, bs-0678R) at 4°C for 12 hours.

Techniques: Methylation, Flow Cytometry

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: Epigenetic programming of μ-opioid receptor gene in mouse brain is regulated by MeCP2 and brg1 chromatin remodelling factor

doi: 10.1111/j.1582-4934.2008.00535.x

Figure Lengend Snippet: Interaction of MeCP2 with the chromatin-remodelling factor, Brg1. (A) – Antibody against Brm was used to immunoprecipitate and visualize the interaction with MeCP2 (detected with anti-MeCP2 antibody in the Western blot) in mouse brain nuclear extracts. Conversely, MeCP2 antibody was used to detect interactions with Brm (detected with anti-Brm antibody in the Western blot). Using MOR-positive differentiated P19 cells (AP4d), MeCP2 antibody was used to immunoprecipitate Brg1 chromatin-remodelling factor. The interaction of MeCP2 with corepressor was also analysed using mSin3A to validate the coimmunoprecipitation. SN: protein supernatant after immunoprecipitation; IP Ab: immunoprecipitation antibody; IB Ab: immunoblotting antibody. (B) – The expression of Brg1 was assessed by Western blot analysis using anti-Brg1 antibody in different cell types/conditions and mouse brain (MB). Brg1 proteins are indicated with their molecular weights: full-length (200 kD); isoforms (75 and 70 kD), consistent with Upstate antibody information for anti-Brg1 (Upstate, 07–478). (C) – ChIP analysis by real-time qPCR of Brg1 interaction. Primers specific for PP-MOR (S-408 and AS-285; Table ) and DP-MOR (S-731 and AS-623) were used to amplify by real-time qPCR genomic DNA sequences immunoprecipitated with anti-Brg1 antibody. Five brain regions (OB, STM, HPT, CRB, and PCX) and NS20Y cells were used for the ChIP analysis. (D) – ReChIP analysis by real-time qPCR. The primers specific for PP-MOR, DP-MOR, and β-actin were the same as those used above. Three brain tissues, STM ( i.e. a site of intermediate MOR expression), HPT (a high MOR site), and CRB (a non-MOR expressing site) were used for the ReChIP. Data are normalized against the input and are the mean ± S.E.M. from three independent experiments. ( Top panel ) first antibody: anti-Brg1, second antibody: anti-MeCP2. ( Centre panel ) First antibody: anti-MeCP2, second antibody: anti-Brg1. ( Bottom panel ) first antibody: anti-Dnmt1, second antibody: anti-MeCP2.

Article Snippet: Pre-cleared lysates (100 μl) diluted in immunoprecipitation buffer (Upstate) were immunoprecipitated overnight at 4°C with 2 μg of antibodies against each of the following: anti-AcH3 (06–599), anti-AcH4 (06–866), anti-H3 dm K4 (07–030), anti-H3 dm K9 (07–441) (all from Upstate), anti-Dnmt1 (Imgenex; IMG-261A), anti-Dnmt3b (Imgenex; IMG-184A) and anti-MeCP2.

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Epigenetic programming of μ-opioid receptor gene in mouse brain is regulated by MeCP2 and brg1 chromatin remodelling factor

doi: 10.1111/j.1582-4934.2008.00535.x

Figure Lengend Snippet: A proposed molecular mechanism for MOR gene regulation. MeCP2 and Dnmt1 bind to hypermethylated DNA form a histone-associated repressor complex, silencing the MOR gene in cerebellar tissue. In the cerebellum, hypermethylation of CpGs around the proximal promoter (PP) coincides with increased interactions with MeCP2 and Dnmt1. This might lead to compaction of the chromatin structure after histone modifications, followed by silencing of the MOR gene in these cells. In striatal cells, intermediate methylation of CpGs around the PP begins as MeCP2 start to dissociate and Brg1 is recruited, concurrent with histone modifications, resulting in intermediate levels of MOR expression. In the hypothalamus, nearly complete demethylation of the CpGs around the PP is observed as MeCP2 dissociates and Brg1 is recruited. Hyperacetylation of histones also occur in the promoter, suggesting active transcription of the MOR gene in the hypothalamic tissue. The components for active transcription shown in the figure, i.e. GTF (general transcription factors) and their associated-RNA polymerase II (Pol II), are putative factors for general genes.

Article Snippet: Pre-cleared lysates (100 μl) diluted in immunoprecipitation buffer (Upstate) were immunoprecipitated overnight at 4°C with 2 μg of antibodies against each of the following: anti-AcH3 (06–599), anti-AcH4 (06–866), anti-H3 dm K4 (07–030), anti-H3 dm K9 (07–441) (all from Upstate), anti-Dnmt1 (Imgenex; IMG-261A), anti-Dnmt3b (Imgenex; IMG-184A) and anti-MeCP2.

Techniques: Methylation, Expressing

Journal: European Journal of Histochemistry : EJH

Article Title: Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells: Role of epigenetic modifications to the inflammation

doi: 10.4081/ejh.2017.2826

Figure Lengend Snippet: Immunofluorescence. Cells grown on coverslips were fixed and stained with the indicated primary and secondary antibodies and DAPI, and subjected to confocal microscopy. NF-kB expression in hPDLSCs (A1) and in hPDLSCs treated with LPS-G (A2). p300 expression in hPDLSCs (B1) and in hPDLSCs treated with LPS-G (B2). DNMT1 expression in hPDLSCs (C1) and in hPDLSCs treated with LPS-G (C2). Data are representative of three independent experiments. Magnification: 63x.

Article Snippet: Sheets were saturated for 120 min at room temperature in blocking buffer (1xTBS, 5% milk, 0.1% Tween-20), then incubated overnight at 4°C in blocking buffer containing primary antibodies to NFkB (1:2000, rabbit) (OriGene), DNMT1 (1:1000, rabbit) (OriGene), p300 (1:750, rabbit) (OriGene) and β-actin (1:750, mouse) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing

Journal: European Journal of Histochemistry : EJH

Article Title: Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells: Role of epigenetic modifications to the inflammation

doi: 10.4081/ejh.2017.2826

Figure Lengend Snippet: Western blot analysis of NF-κB (A), NF-B in nuclear extract (B), DNMT1 (C) and p300 (D) expression in hPDLSCs and in hPDLSCs treated with LPS-G for 24 h. -actin was used as a housekeeping protein (E). Densitometric analysis of protein specific bands (F). **P<0.01; ***P<0.001.

Article Snippet: Sheets were saturated for 120 min at room temperature in blocking buffer (1xTBS, 5% milk, 0.1% Tween-20), then incubated overnight at 4°C in blocking buffer containing primary antibodies to NFkB (1:2000, rabbit) (OriGene), DNMT1 (1:1000, rabbit) (OriGene), p300 (1:750, rabbit) (OriGene) and β-actin (1:750, mouse) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: European Journal of Histochemistry : EJH

Article Title: Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells: Role of epigenetic modifications to the inflammation

doi: 10.4081/ejh.2017.2826

Figure Lengend Snippet: Gene expression. Next generation sequencing demonstrated the modulation of genes expressed in untreated and LPS-G treated hPDLSC. P300, TNFAIP1, IL6ST and HDAC2 were expressed greater than 2-fold (Log2 fold change; Q<0.05). DNMT1 and HDAC1 genes were significantly downregulated in LPS-G stimulated hPDLSCs when compared with untreated cells (Q<0.05). Up-regulated transcripts are highlighted in red color; downregulated transcripts are highlighted in green color.

Article Snippet: Sheets were saturated for 120 min at room temperature in blocking buffer (1xTBS, 5% milk, 0.1% Tween-20), then incubated overnight at 4°C in blocking buffer containing primary antibodies to NFkB (1:2000, rabbit) (OriGene), DNMT1 (1:1000, rabbit) (OriGene), p300 (1:750, rabbit) (OriGene) and β-actin (1:750, mouse) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Next-Generation Sequencing