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: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: A. Western blot was performed to check the expression levels of ERα and DNMTs in MCF-7 cells transfected with ERα expression vectors. B. Luciferase reporter assay showed that over-expression of ERα enhanced the promoter activities of DNMT1 and DNMT3b, but not DNMT3a, in MCF-7 cells. C. Real-time PCR showed that over-expression of ERα up regulated the intracellular mRNA levels of DNMT1 and DNMT3b, but not DNMT3a, in MCF-7 cells. D. Western blot was performed to check the expression levels of ERα and DNMTs in MCF-7/PTX cells transfected with ERα-shRNA plasmids. E. Luciferase reporter assays showed that knockdown of ERα reduced DNMT1 and DNMT3b promoter activities in MCF-7/PTX cells. F. Real-time PCR showed that knockdown of ERα reduced the DNMT1 and DNMT3b intracellular mRNA levels in MCF-7/PTX cells.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Transfection, Luciferase, Reporter Assay, Over Expression, Real-time Polymerase Chain Reaction, shRNA, Knockdown

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: A. Diagram of the ERα binding sites in the human DNMT1 and DNMT3b gene promoters indicated by bioinformatics analysis. B. ChIP assay revealed that the DNMT1-S2 and DNMT1-S3 and the DNMT3b-S1 and DNMT3b-S3 were immunoprecipitated with ERα antibody, confirming ERα binds to theses sequences in breast cancer cells. C. qChIP assay indicated that the bindings of ERα to the DNMT1 and DNMT3b promoters were significantly increased in MCF-7/PTX cells when compared with the parental MCF-7 cells. D. The qChIP assay was repeated in ZR-75-1/PTX cells and similar results were obtained, indicating that anticancer drug exposure enhanced binding of ERα to the DNMT1 and DNMT3b promoters.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Binding Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: A. Western blot analysis of DNMT1 expression in MCF-7 cells transiently transfected with DNMT1 expression plasmids (upper panel). MTT assay indicated that over-expression of DNMT1 increased viability of breast cancer cells under the stress of PTX treatment (lower panel). B. Similar experiments were performed to test the effect of DNMT3b on the response of breast cancer cells to PTX. DNMT3b over-expression increased the cell survival in the presence of PTX. C, D. Western blot analysis of DNMT1 expression in MCF-7/PTX (upper left) or ZR-75-1/PTX (upper right) cells transiently transfected with DNMT1-shRNA plasmids (upper). MTT assay was performed to determine cell viabilities of MCF-7/PTX (lower left) or ZR-75-1/PTX cells (lower right) treated with PTX at different concentrations. E, F. Kaplan-Meier analysis revealed negative correlation between DNMT1 and RFS and DMFS of ERα-positive breast cancer patients. G, H. Kaplan-Meier analysis displayed the similar results regarding the correlation between DNMT3b and the RFS and DMFS.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Transfection, MTT Assay, Over Expression, shRNA

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: A, B. Western blot was performed to determine the ERα knockdown efficiency in MCF-7/PTX (upper left) or ZR-75-1/PTX cells (upper right) transfected with ERα-shRNA. MTT assay showed that knockdown of ERα partly restored the sensitivity of PTX drug resistant breast cancer cells (lower panel), indicating ERα contributed to breast cancer drug resistance. C, D. Western blot was performed to check the transfection efficiencies in MCF-7 cells transfected with ERα expression plasmids together with either DNMT1-shRNA plasmids (C, upper panel) or DNMT3b-shRNA plasmids (D, upper panel). MTT assays were performed to examine the cell viability. Knockdown of DNMT1 (C, lower panel) or DNMT3b (D, lower panel) reduced the effect of ERα over-expression on the drug resistance of the MCF-7 cells. E. MTT assays showed that double knockdown of DNMT1 and DNMT3b restricted the effect of ERα over-expression on drug resistance more than the single DNMT knockdown.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Western Blot, Knockdown, Transfection, shRNA, MTT Assay, Expressing, Over Expression

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: A. Luciferase reporter assay was performed to detect DNMTs promoter activities following treatment with graded concentrations of estrogen. B. Real-time PCR was performed to detect transcriptional levels of DNMTs in MCF-7 cells treated with estrogen. C. Western blot was performed to detect the expression levels of DNMT1 and DNMT3b in MCF-7 cells treated with estrogen. D. qChIP assay confirmed that estrogen treatment increased the bindings of ERα to DNMT1 and DNMT3b promoters in MCF-7 cells. E–J. MTT assay showed that estrogen increased the resistance of MCF-7 and ZR-75-1 cells to multi anticancer drugs, including PTX (E, H), EPI (F, I) and VCR (G, J).

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, MTT Assay

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: Quantitative methylation-sensitive PCR (qMSP) was performed to detect the genomic DNA methylation levels. A. qMSP showed that global methylation level was increased in MCF-7 cells transfected with ERα expression plasmids and was decreased in MCF-7/PTX cells transfected with ERα-shRNA plasmids when compared with their controls. B. qMSP showed that global methylation level was increased in MCF-7 cells transfected with DNMT1 expression plasmids compared with the control. C. qMSP showed that knockdown of DNMT1 in MCF-7 cells significantly restrained the ERα-induced global hypermethylation. D, E. qMSP showed that over-expression or knockdown of DNMT3b had no detectable effect on global DNA methylation level.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Methylation, DNA Methylation Assay, Transfection, Expressing, shRNA, Control, Knockdown, Over Expression

Journal: Oncotarget

Article Title: ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells

doi: 10.18632/oncotarget.8038

Figure Lengend Snippet: Representative immunohistochemical staining pictures of ERα, DNMT1 and DNMT3b in breast cancer tissues. The upper panel represented the strong positive staining and the lower panel represented the weak positive staining. The level of ERα in breast cancer tissues showed a statistically positive correlation with DNMT1, while no significant correlation with DNMT3b was observed.

Article Snippet: Antibodies against DNMT1 were purchased from Cell Signaling Technology, anti-DNMT3a was purchased from Santa Cruz, anti-DNMT3b was obtained from Abcam, and anti-ERα was from Santa Cruz; anti-β-actin was obtained from Sigma-Aldrich.

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

doi: 10.1016/j.omtn.2023.102031

Figure Lengend Snippet: Nim inhibits DNMTs expression and restores SFRP1 expression in BCSCs (A) Boxplot showing the expression of SFRPs in breast cancer patients (GEPIA 2 database). The y axis log scale is represented as log 2 (TPM + 1), and the method for differential analysis is the one-way ANOVA, using disease state (tumor or normal) as the variable for calculating differential expression and statistical significance, with each dot representing a distinct tumor or normal sample. Red boxplot, tumor tissues (n = 1085); blue boxplot, normal tissues (n = 291). TPM, transcript per million. (B) The correlation between the gene expression level of SFRP1 and methylation rate of SFRP1 promoter from TCGA database, obtained by cBioportal Browser. (C) Real-time PCR assay showing mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1 , DNMT3A , and DNMT3B upon treatment with Nim, Nim NPs, and DAC. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (D) Immunoblotting analysis demonstrating protein expression of DNMTs following treatment with Nim, Nim NPs, and DAC (5 μM) respectively in BCSCs. (E) Real-time PCR assay showing mRNA expression of SFRP1 upon treatment with Nim, Nim NPs, and DAC. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (F) Immunoblotting analysis demonstrating protein expression of SFRP1 following treatment with Nim, Nim NPs, and DAC. (G and H) pcDNA3/Myc-DNMT1 and pcDNA3/Myc-DNMT3A were transiently overexpressed and cells were treated with Nim or Nim NPs (5 μM) for 48 h and subjected to immunoblotting with indicated antibodies respectively. (I–K) Bar graph demonstrates quantification of methylation level at different regions of SFRP1 CpG island (SCpGR1, SCpGR2, and SCpGR3) in bisulfite-converted DNA with or without treatment with Nim, Nim NPs, or DAC (5 μM) measured by methylation-specific PCR (MSP) using methylation and unmethylation-specific primers. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. SCpGR1, SFRP1 CpG island region 1; SCpGR2, SFRP1 CpG island region 2; SCpGR3, SFRP1 CpG island region 3.

Article Snippet: The sheared chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and soluble chromatin was incubated overnight with blocking using 5% BSA followed by 15 μL of magnetic beads (Pierce ChIP-grade Protein A/G Magnetic Beads, Thermo Fisher Scientific, 26162) and 1 μg of antibody against DNMT1 (Thermo Fisher Scientific, PA5120552), and DNMT3A (Thermo Fisher Scientific, PA1882), and immunoglobulin (Ig) G as a negative control (Diagenode, C15410206).

Techniques: Expressing, Methylation, Real-time Polymerase Chain Reaction, Comparison, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

doi: 10.1016/j.omtn.2023.102031

Figure Lengend Snippet: Nim inhibits SFRP1 promoter hypermethylation and consequently downregulates Wnt/β-catenin signaling in BCSCs (A) Schematic representation of SFRP1 gene locus and regions analyzed before and after Nim or Nim NP treatment. The solid box (green) represents SFRP1 CpG island, lines (red) represent the regions evaluated to estimate methylation status before and after treatment of Nim or Nim NPs compared to the positive control DAC, and lines (blue) represent negative controls selected from upstream and downstream of non-CpG region. (B–I) Chromatin immunoprecipitation (ChIP)-qPCR represents DNMT (DNMT1 and DNMT3A) enrichment in different CpG island regions such as transcription start site (TSS 1–6) and promoter (PRM 7–8) of the SFRP1 gene before and after treatment with Nim, Nim NPs, and DAC (5 μM) respectively in BCSCs. IgG pull-down was used as the control. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (J) Immunoblotting analysis demonstrating protein expression of Wnt/β-catenin signaling associated markers following treatment with Nim, Nim NPs, and DAC (5 μM), respectively, in BCSCs.

Article Snippet: The sheared chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and soluble chromatin was incubated overnight with blocking using 5% BSA followed by 15 μL of magnetic beads (Pierce ChIP-grade Protein A/G Magnetic Beads, Thermo Fisher Scientific, 26162) and 1 μg of antibody against DNMT1 (Thermo Fisher Scientific, PA5120552), and DNMT3A (Thermo Fisher Scientific, PA1882), and immunoglobulin (Ig) G as a negative control (Diagenode, C15410206).

Techniques: Methylation, Positive Control, Chromatin Immunoprecipitation, Comparison, Western Blot, Expressing

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