Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 3 PPI decreased protein expression of DNMT1 and EZH2 through SAPK/JNK pathway. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot. b PC9 and A549 cells were exposed to PPI (1.6 μM) for 24 h, followed by measuring the mRNA levels by qRT-PCR. c PC9 and A549 cells were transfected with a wild type human EZH2 promoter reporter construct ligated to luciferase reporter gene and internal control for 24 h, followed by treating with PPI for an additional 24 h. Afterwards, the promoter activities were determined using the Secrete-Pair Dual Luminescence Assay Kit as described in the Materials and Methods Section. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. d PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot using antibodies against EZH2 and DNMT1. The bar graphs represent the mean ± SD of EZH2 or DNMT1/GAPDH of three independent experiments. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Expressing, Concentration Assay, Western Blot, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control, Luminescence Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 4 Activation of SAPK/JNK pathway-mediated inhibition of p65 expression contributed to the PPI-decreased protein expression of DNMT1. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h, followed by measuring the protein expression of p65 and p50 by Western blot. The bar graphs represent the mean ± SD of p65/GAPDH of three independent experiments. b PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of phosphorylation of SAPK/JNK and p65 protein were detected by Western blot. c PC9 and A549 cells were transfected with control and p65 overexpression vector for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, p65 and DNMT1 protein expressions were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Activation Assay, Inhibition, Expressing, Concentration Assay, Western Blot, Phospho-proteomics, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 5 Exogenously expressed EZH2 not only restored cell growth, but also feedback antagonized PPI increased SAPK/JNK signaling. a PC9 and A549 cells were transfected with the control or expression constructs of DNMT1 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression were determined using Western blot. b–c PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression (b) and cell viability (c) were determined using Western blot and MTT assays, respectively. d PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2, phosphor-SAPK/JNK were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Transfection, Control, Expressing, Construct, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 6 Effects of PPI in subcutaneous xenograft model. Mice (n = 12/group) were divided to 3 groups [Con (saline), Low (L, 1 mg/kg) and High (H, 3 mg/kg)], and PPI was given by intraperitoneal injection for up to 27 days. a The xenografts were assessed by in vivo bioluminescence imaging at the sixth and end of the experiments (on day 6 and 27). The tumor growth was monitored by injecting luciferin in the mice followed by measuring bioluminescence and analyzed as described in the Materials and Methods section. Representative images are shown. b and c The xenografts were harvested on day 27, and the size and weight of tumors were determined. The bar graphs represented the tumor weight and size of mice results of as mean ± SD. d–e At the end of the experiments, xenografted tumors were isolated from individual animals and the corresponding lysates were detected p-SAPK/JNK, DNMT1, p65 and EZH2 proteins by Western blot and Immunohistochemistry as described in the Materials and Methods sections. Scale bar 50 μM. GAPDH was used as loading control for Western blot. Values in bar graphs were given as the mean ± SD from three independent Western blot experiments. *Indicates the significant difference from untreated control (p < 0.05). f The diagram shows that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Saline, Injection, In Vivo, Imaging, Isolation, Western Blot, Immunohistochemistry, Control, Inhibition, Gene Expression

Journal: bioRxiv

Article Title: Establishment of beta cell heterogeneity via differential CpG methylation at Nnat

doi: 10.1101/2023.02.04.527050

Figure Lengend Snippet: ( A ) Representative confocal microscopy of pancreatic cryosections from Nnat WT/eGFPpat mice at embryonic (E) day 17.5 and postnatal (P) day 7 and 14 (n = 15 and 14 mice, respectively). Sections were immunostained with antibodies against endogenous neuronatin (NNAT, green) and DNMT3A or DNMT1 (both grey). Nuclei are visualised with DAPI. Scale bar = 50μm. ( B ) Representative confocal microscopy of pancreatic cryosections from mice with conditional deletion of DNMT3A under the control of the Pdx1 promoter ( Pdx1-Cre + Dnmt3a fl/fl vs control ( Pdx1-Cre − Dnmt3a fl/fl mice at postnatal (P) day 6. Sections were immunostained with antibodies against endogenous neuronatin (NNAT, red) and insulin (INS, green). ( C ) Quantification of NNAT + beta cells from images shown in B, expressed as NNAT/INS co-positive cells as a percentage of total INS-positive cells. Nuclei are visualised with DAPI. Scale bar = 50μm (n = 8 islets from two mice per genotype, unpaired Students t test, ** P < 0.01).

Article Snippet: Sections were immunostained using primary antibodies diluted in blocking buffer against NNAT (clone EPR13554(B), ab181353 from Abcam, 1:1000), GFP (GFP-1010, Aves Labs, 1:3000) INS (I2018, Sigma-Aldrich, 1:5000), somatostatin (SST) (American Research Products, 13-2366, 1:1000), GCG (clone C-18, sc-7779, Santa Cruz Biotechnology, 1:50), DNMT1 (60B1220.1, Novus Biologicals, 1:500) and DNMT3A (64B1446, Novus Biologicals, 1:500) with overnight incubation at 4°C followed by incubation with secondary antibody (Alexa Fluor, Thermo Fisher Scientific, 1:500).

Techniques: Confocal Microscopy, Control

Journal: NPJ Precision Oncology

Article Title: Loss of MicroRNA-29b promotes DNMT3b-mediated STING downregulation to attenuate radiotherapy-induced antitumor immunity in KRAS-mutated colorectal cancer

doi: 10.1038/s41698-026-01290-8

Figure Lengend Snippet: A WiDr cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. WiDr-Vec. and WiDr-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. B CoLo320 cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. CoLo320-Vec. and CoLo320-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. C HCT116 cells were infected with lentivirus carrying pLKO-scramble shRNA (shNC) or pLKO-shKRAS (shKRAS) and selected for three days. HCT116 shNC and HCT116 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. D SW620 shNC and SW620 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. E WiDr and Colo320DM (endogenous wild-type KRAS) cells were individually infected with vector (Vec.) and KRAS G12D . Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. F CT26, HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shNC and shKRAS. Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. G KRAS G12D -tranduced WiDr (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. H . KRAS G12D -tranduced CoLo320 (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. I HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shDNMT1, shDNMT3a and shDNMT3b. Cells were selected by puromycin for three days. The mRNA level of STING was evaluated by qRT-PCR ( n = 3). One-way ANOVA t test. * p < 0.05. J The protein level of STING was evaluated by immunoblotting ( n = 3).

Article Snippet: The following antibodies were used: phospho-STING-S365 (#19781, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), cleaved caspase-3 (#9661, Cell Signaling Technology and IR96–401, iReal Biotech.), calreticulin (#12238, Cell Signaling Technology), beta-actin (Ab8227, Abcam), DNMT1 (#5032, Cell Signaling Technology), DNMT3a (#49768, Cell Signaling Technology), DNMT3b (#57868, Cell Signaling Technology), KRAS (A1190, ABclonal) and GAPDH (IR3-8, iReal Biotech.)

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

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 the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 1. The epigenetic factors DNMT1 and MeCP2 and the transcriptional factor REST are involved in the negative modulation of Ncx1 mRNA and protein expression. A and B, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y cells transfected for 48 hours with siDNMT1 or siMeCP2 at a final concentration of 50 nM. An siCTL was used as scrambled. *P≤0.05 vs siCTL by Student’s t test (n=3). C, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hours with siCTL, siDNMT1, siMeCP2, and siREST. *P≤0.05 vs siCTL by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). D and E, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y transfected for 48 hours with vector overexpressing DNMT1 or MeCP2. pcDNA3.1 vector was used as EV. *P≤0.05 vs EV by Student’s t test (n=3). F, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hour with EV, DNMT1, MeCP2, or REST plasmids. *P≤0.05 vs EV by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). CTL indicates control; DNMT1, DNA-methyltransferase-1; EV, empty vector; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; si, small interfering; and siCTL, control siRNA.

Article Snippet: The following small interfering RNAs (siRNAs) have been used: mouse DNMT1 (sc 35 203, Santa Cruz Biotechnology); mouse MeCP2, HDAC1, HDAC2, NCX1 (SI00195594, SI02732982, SI00173551, SI01424920 QIAGEN); mouse REST (EMU029201, SIGMA); and human DNMT1, REST (SI02663409, SI02929693, QIAGEN); and human MeCP2 (EMU030271, SIGMA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay, Plasmid Preparation, Control, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 4. DNMT1 and MeCP2 bind Ht, but not Br promoter sequence, in the temporoparietal cortex 24 hours after tMCAO. Chromatin immunoprecipitation with anti-DNMT1 (A), anti-MeCP2 (B) anti-REST (C), anti-HDAC1 (D), anti-HDAC2 (E), antibodies followed by qPCR of Ncx1 brain promoter (Br) (white columns) and Ncx1 heart promoter (Ht) (gray columns) in peri-ischemic cortex of mice euthanized 12 or 24 hours after tMCAO. IgG was used as negative control. *P≤0.05 vs Sham immunopreciptated with IgG antibody by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3). DNMT1 indicates DNA-methyltransferase-1; HDAC, histone deacetylase; IP, immunoprecipitation; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; and tMCAO, transient middle cerebral artery occlusion.

Article Snippet: The following small interfering RNAs (siRNAs) have been used: mouse DNMT1 (sc 35 203, Santa Cruz Biotechnology); mouse MeCP2, HDAC1, HDAC2, NCX1 (SI00195594, SI02732982, SI00173551, SI01424920 QIAGEN); mouse REST (EMU029201, SIGMA); and human DNMT1, REST (SI02663409, SI02929693, QIAGEN); and human MeCP2 (EMU030271, SIGMA).

Techniques: Sequencing, Chromatin Immunoprecipitation, Negative Control, Histone Deacetylase Assay, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 1. Schematic diagram depicting Dnmt1 (a) and Lox (b) promoter. a, exon representing the somatic transcription start (marked with 1) is repre- sented as small open box (untranslated region) and large black arrow (translated region). The three putative FLI1-binding sites are shown as gray boxes. Forward (fw) cloning primers annotated with the resulting amplicon size and reverse (rv) cloning primer are marked as white arrows. Binding sites of amplifi- cation primers for Fli1 ChIP analysis are marked as black arrows. The numbers below the gene line denote the distance to the transcription start. b, represen- tation of Lox proximal promoter and the first exon showing the fragment used for methylation analysis (gray line). Lox genomic structure shows a part of the first exon (475 bp), including 374 bp of the 5-untranslated region (open box) and the first 101 bp of the coding region (black arrow). To generate a suita- ble fragment (gray line) for methylation analysis by “Methyl Miner” assay, DNA was restricted by MboII restriction enzyme (M). Forward (fw) and reverse (rv) primers for subsequent amplification by qRT-PCR are marked as white arrows. The vertical bars indicate the CpGs in the DNA strand, and the numbers below the gene line denote the distance to the transcription start.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Binding Assay, Cloning, Amplification, Methylation, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 3. Fli1 (a) and Dnmt1 (b) mRNA expression after treatment of MC3T3-E1 cells with 30 ng/ml recombinant murine IL-6 for 36 and 48 h. Com- pared with untreated control (Co), Fli1 mRNA expression is significantly up-regulated after 36 h of IL-6 treatment, whereas Dnmt1 mRNA expression remains unaffected at this time. After 48 h, 12 h after Fli1 stimulation, Dnmt1 also shows a significant increase in mRNA expression by IL-6 treatment. At this time, Fli1 mRNA expression is still increased by IL-6; however, the regulation is not significant. As shown in c and d, treatment of the cells with increasing concen- trations of IL-6 at the times showing significant mRNA stimulation for Fli1 and Dnmt1 revealed a significant up-regulation for both genes already at 3 ng/ml IL-6 in culture medium. The up-regulative effect of the cytokine was already reached at this concentration saturation for both genes. To analyze mRNA ex- pression of Fli1 and Dnmt1, RNA was isolated and analyzed by qRT-PCR. Gene expression was normalized to 18 S rRNA. Treated probes are referred as fold change to untreated control; *, p 0.05; **, p 0.01; ***, p 0.001. For all graphs n 3.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Expressing, Recombinant, Control, Concentration Assay, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 4. a, as measured by reporter gene assay, Fli1-binding sites/promoter length correlated with the IL-6-dependent activation of the Dnmt1 promoter. A significant increase in promoter activity by IL-6 was seen when the putative FLI1-binding sites at 60 bp (190-bp fragment) and at 96 bp (220-bp frag- ment) from the somatic transcriptional start site were included in the Dnmt1 promoter. Point mutation at one or more putative FLI1-binding sites in the 220-bp Dnmt1 promoter fragment reduced IL-6 (30 ng/ml) responsiveness of the Dnmt1 promoter. Mutation at all putative FLI1 sites decreased significantly IL-6-dependent Dnmt1 promoter activation (b). Promoter activity was measured by SEAP2 reporter gene assay. Values are represented as mean S.D., and untreated controls are set to 0 in a and to 1 in b. b depicts one representative result of total three experiments. Treated probes are referred as fold change to nontreated control (NT); *, p 0.05; **, p 0.01; ***, p 0.001. b, represents significant differences to IL-6 treated, nonmutated Dnmt1 vector (NM, white bar): , p 0.05; , p 0.01; , p 0.001. a, n 3; b, n 4.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Reporter Gene Assay, Binding Assay, Activation Assay, Activity Assay, Mutagenesis, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 7. mRNA expression of Dnmt1, a gene involved in DNA methyla- tion, is enhanced by Hcys in MC3T3-E1 cells. As demonstrated in a, when compared with untreated control (Co), mRNA expression of the DNA methyl- ation-related gene Dnmt1 was significantly enhanced by 3.6 mM Hcys after 6 and 14 days (d) of treatment. Hcys-dependent up-regulation of DNMT1 was confirmed at the protein level after 6 days of treatment (b and c). To analyze mRNA expressions, RNA was isolated and analyzed by qRT-PCR. Gene expression was normalized to 18 S rRNA. 30 g of protein of each sample were fractionated by SDS-gel electrophoresis and immunoblotted. Values are represented as mean S.D.; untreated control (Co) is set to 1, and treated probes are referred as fold change to control. *, p 0.05; ***, p 0.001; for all graphs n 3. One representative immunoblot is shown. Lane M refers to protein molecular weight standards.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Expressing, Control, Isolation, Quantitative RT-PCR, Gene Expression, SDS-Gel, Electrophoresis, Western Blot, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 10. Hcys-mediated up-regulation of Dnmt1 is IL-6- and Fli1-dependent. After 1 week of Hcys treatment, IL-6 Ab significantly abrogated the ef- fect of Hcys on Dnmt1 expression while leaving Dnmt1 mRNA expression in Hcys-untreated cells unaffected (a). Involvement of FLI1 transcription factor in Hcys-driven Dnmt1 up-regulation was shown by chromatin immunoprecipitation analysis (ChIP) (b). After 1 week, Hcys-treated cells showed a significant increase of FLI1 binding affinity to the selected Dnmt1 promoter sequence. mRNA expressions as well as ChIP-DNA were analyzed by qRT-PCR. Gene expres- sion was normalized to 18 S rRNA. For ChIP quantitation, DNA signals of the FLI1-precipitated chromatin were normalized to the unprecipitated chromatin fraction (1% of the total chromatin). Values are represented as mean S.D.; untreated control (Co) is set to 1, and treated probes are referred as fold change to control. *, p 0.05; , p 0.01 refers to significance to Hcys-treated probes; for all graphs n 3.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Expressing, Chromatin Immunoprecipitation, Binding Assay, Sequencing, Quantitative RT-PCR, Quantitation Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

doi: 10.1074/jbc.m110.166181

Figure Lengend Snippet: FIGURE 13. Schematic representation of the suggested pathway for the down-regulation of Lox by Hcys. Hcys stimulates IL-6 expression, which in turn activates JAK2 kinase (IL-6 as well as JAK2 kinase can be blocked by an IL-6 Ab as well as by the inhibitor AG490, respectively), leading to an up- regulation of the Dnmt1 transcription factor Fli1. This increases Dnmt1 expression, which provokes promoter methylation of Lox and thus its tran- scriptional depletion (co-treatment of the cells with the DNA (cytosine-5)- methyltransferase (Dnmt) inhibitor zebularine abrogates Hcys Lox promoter methylation). Consequently, the post-translational collagen cross-link for- mation is reduced. Abbreviations used are as follows: Hcys, homocysteine; IL-6, interleukin 6; IL-6 Ab, interleukin 6 antibody; IL-6R, interleukin 6 recep- tor; Fli1, Friend leukemia virus integration 1; Dnmt1, DNA methyltransferase 1; Lox, lysyl oxidase; CH3, methyl group.

Article Snippet: Subsequently, the filters were incubated for 1 h at room temperature with antibodies against FLI1 ((C19), HELLS (Lsh, H-240)), LOX (H-140), DNMT1 (K-18, all Santa Cruz Biotechnology) diluted 1:200 in blocking buffer.

Techniques: Expressing, Methylation, Virus

Journal: The FEBS journal

Article Title: MCPH1 maintains long-term epigenetic silencing of ANGPT2 in chronic lymphocytic leukemia.

doi: 10.1111/febs.13245

Figure Lengend Snippet: Fig. 5. Physical interactions between MCPH1 and DNMT1, DNMT3b and E2F1 based on the co-IP method. In the left panel, the gels represent the MCPH1 pulldown expression levels of the respective immunoprecipitation (IP) antibodies in nuclear extracts of MCF-7 cells and three CLL cell lines (HG3, RAMOS, and Mec1). The total protein level of the antibody used for IP is shown as a separate lane below each MCPH1 blot. On the right side, the total protein expression levels, as determined in total cell lysates, of all of the antibodies used in co-IP are shown. b-Actin and GAPDH were used as internal loading controls. Ab, antibody; FL, full-length; WB, western blot.

Article Snippet: The antibodies used were mAb against MCPH1 (MA5-18057; Thermoscientific, Rockford, USA), polyclonal antibody against E2F1 (ab4070; Abcam, Cambrige, UK), mAb against DNMT1 (IMG-261A; Imgenex, USA), polyclonal antibody against DNMT3b (pAB-076-005; Diagenode, Liege, Belgium), and IgG (negative control; OneDay ChIP Kit).

Techniques: Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Western Blot