human dnmt3a catalytic domain  (Addgene inc)

Journal: Food Science & Nutrition

Article Title: Flavonoids in Rosa roxburghii Tratt Fermentation Broth Ameliorate Obesity via DNMT3a / SIRT1 ‐Mediated Epigenetic Modulation

doi: 10.1002/fsn3.70892

Figure Lengend Snippet: Changes in DNA methylation after intervention with RRTFB ( n = 3). (A) ELISA detection of 5‐MC and DNMT3a levels in liver, epididymal adipose tissue, and testicular tissue. (B) Immunohistochemical analysis of DNMT3a in epididymal adipose tissue. The small image on the left is 100×, and the large image on the right is 400×. (C) Immunohistochemical average optical density value. (D) Methylation ratio by DNA methylation sequencing. (E) Methylation rate of chromosome. * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001.

Article Snippet: All solutions were subjected to filtration through a 0.22 μm Millipore filter. pH 4.0 recombinant human DNMT3A protein (Full Length, GST Tag) (D353‐380G; Sino Biological, Sweden) was immobilized on the sensor chip surface through amine groups, and fisetin (HY‐N0182; MCE, USA) was injected for 1:1 binding, and the affinity between fisetin and DNMT3A was tested.

Techniques: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Methylation, Sequencing

Journal: Food Science & Nutrition

Article Title: Flavonoids in Rosa roxburghii Tratt Fermentation Broth Ameliorate Obesity via DNMT3a / SIRT1 ‐Mediated Epigenetic Modulation

doi: 10.1002/fsn3.70892

Figure Lengend Snippet: Molecular mechanisms of SIRT1 regulation in epididymal adipose tissue. (A) Immunofluorescence co‐localization of SIRT1 (green) and PPARγ (red) with nuclear DAPI staining (blue) (400×). (B) WB analysis of PPARγ, NF‐κB, and HIF1a protein expression. (C) mRNA expression levels of PPARγ, NF‐κB, and HIF1a in epididymal adipose tissue. (D) Validation of DNMT3a overexpression and its effect on SIRT1 protein levels in 3T3‐L1 adipocytes. (E) ChIP analysis of DNMT3a binding to the SIRT1 promoter region. p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: All solutions were subjected to filtration through a 0.22 μm Millipore filter. pH 4.0 recombinant human DNMT3A protein (Full Length, GST Tag) (D353‐380G; Sino Biological, Sweden) was immobilized on the sensor chip surface through amine groups, and fisetin (HY‐N0182; MCE, USA) was injected for 1:1 binding, and the affinity between fisetin and DNMT3A was tested.

Techniques: Immunofluorescence, Staining, Expressing, Biomarker Discovery, Over Expression, Binding Assay

Journal: Food Science & Nutrition

Article Title: Flavonoids in Rosa roxburghii Tratt Fermentation Broth Ameliorate Obesity via DNMT3a / SIRT1 ‐Mediated Epigenetic Modulation

doi: 10.1002/fsn3.70892

Figure Lengend Snippet: Phytochemical characterization of RRTFB and molecular docking analysis with DNMT3a. (A) Chemical classification of metabolites identified in RRTFB. (B) Subclass of prenol lipids. (C) Subclass of fatty acyls. (D) Subclass of flavonoids. (E) Relative abundance of major flavonoid components ( x ‐axis: Compounds; y ‐axis: Normalized peak area). (F) Molecular docking models showing interactions between DNMT3a (cyan) and flavonoids (purple): (−)‐epicatechin, fisetin, morin and DNMT3a. The numerical value in the right figure represents hydrogen bonding strength, yellow represents the binding site, and the amino acids are marked in the small figure above. (G) SPR analysis of fisetin‐DNMT3a binding kinetics ( K d = 14.6 μM).

Article Snippet: All solutions were subjected to filtration through a 0.22 μm Millipore filter. pH 4.0 recombinant human DNMT3A protein (Full Length, GST Tag) (D353‐380G; Sino Biological, Sweden) was immobilized on the sensor chip surface through amine groups, and fisetin (HY‐N0182; MCE, USA) was injected for 1:1 binding, and the affinity between fisetin and DNMT3A was tested.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: The up-regulation of SYNCRIP promotes the proliferation and tumorigenesis via DNMT3A/p16 in colorectal cancer

doi: 10.1038/s41598-024-59575-6

Figure Lengend Snippet: Effect of SYNCRIP on regulating the expression of DNMT3A. ( A – C ) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA (SYN sh) for 72 h, the mRNA and protein level of DNMTs were detected by QRT-PCR ( A , B ) and Western blotting ( C ). * P ˂ 0.05, ** P ˂ 0.01. ( D – F ) SW480 and HCT116 cells were transfected with empty vector (EV) and SYNCRIP plasmid, the mRNA and protein level of DNMTs were detected by QRT-PCR ( D , E ) and Western blotting ( F ). * P ˂ 0.05, ** P ˂ 0.01. ( G ) Expression level of DNMT3A in tumor is higher than in normal tissue, shown by GEPIA database. ( H ) The correlation between SYNCRIP and DNMT3A in colorectal cancer.

Article Snippet: DNMT3A overexpression plasmid was purchased from Origene (#RC208192, MD USA).

Techniques: Expressing, Infection, shRNA, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: The up-regulation of SYNCRIP promotes the proliferation and tumorigenesis via DNMT3A/p16 in colorectal cancer

doi: 10.1038/s41598-024-59575-6

Figure Lengend Snippet: SYNCRIP regulated colorectal cancer cell growth and migration via DNMT3A. ( A ) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the protein level of DNMT3A was detected by Western blotting. ( B , C ) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the proliferation and survival of SW480 and HCT116 cells were measured using CCK-8 assay ( B ) and trypan blue stain ( C ). ** P ˂ 0.01. ( D , E ) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the motility of SW480 and HCT116 cells were analyzed using migration assay ( D ) and wound healing assay ( E ).

Article Snippet: DNMT3A overexpression plasmid was purchased from Origene (#RC208192, MD USA).

Techniques: Migration, Infection, shRNA, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, Wound Healing Assay

Journal: Scientific Reports

Article Title: The up-regulation of SYNCRIP promotes the proliferation and tumorigenesis via DNMT3A/p16 in colorectal cancer

doi: 10.1038/s41598-024-59575-6

Figure Lengend Snippet: SYNCRIP regulated the expression of p16 via DNMT3A. ( A , B ) SW480 and HCT116 cells were infected with scr or DNMT3A shRNA for 72 h, the protein and mRNA level of p16 were detected by Western blotting ( A ) and QRT-PCR ( B ). ** P ˂ 0.01. ( C , D ) SW480 and HCT116 cells were infected with scr or SYNCRIP (shRNA) shRNA followed with or without DNMT3A plasmid, the protein and mRNA level of p16 were detected by Western blotting (C) and QRT-PCR ( D ). ** P ˂ 0.01. ( E ) The correlation between SYNCRIP and p16 in colorectal cancer.

Article Snippet: DNMT3A overexpression plasmid was purchased from Origene (#RC208192, MD USA).

Techniques: Expressing, Infection, shRNA, Western Blot, Quantitative RT-PCR, Plasmid Preparation