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: 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: 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: Cerebral cortex (New York, N.Y. : 1991)

Article Title: The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

doi: 10.1093/cercor/bhw341

Figure Lengend Snippet: Figure 5 Postmitotic Dnmt1 deletion in POA-derived interneurons leads to migratory defects during embryonic development. (A, B) Location and number of Hmx3-Cre/

Article Snippet: For siRNA transfection of N2a cells, reverse lipofection with Lipofectamin 2000 (Thermo Fisher Scientific) was performed with 50 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 465318; Invitrogen), Dnmt1 or Pak6 siRNA (Pak6 siRNA sc44879; Dnmt1 siRNA sc-35203; Santa Cruz Biotechnology) for 5 h in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific).

Techniques: Derivative Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

doi: 10.1093/cercor/bhw341

Figure Lengend Snippet: Figure 6 Dnmt1 deficiency causes morphological abnormalities and impairments of the migratory capacity. (A, B) Inverted microphotographs and drawings of repre-

Article Snippet: For siRNA transfection of N2a cells, reverse lipofection with Lipofectamin 2000 (Thermo Fisher Scientific) was performed with 50 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 465318; Invitrogen), Dnmt1 or Pak6 siRNA (Pak6 siRNA sc44879; Dnmt1 siRNA sc-35203; Santa Cruz Biotechnology) for 5 h in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific).

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

doi: 10.1093/cercor/bhw341

Figure Lengend Snippet: Figure 7 Noncanonical actions of DNMT1 regulate Pak6 expression level affecting the morphology of postmitotic POA cells. (A) Schematic illustration of the region

Article Snippet: For siRNA transfection of N2a cells, reverse lipofection with Lipofectamin 2000 (Thermo Fisher Scientific) was performed with 50 nM control siRNA (BLOCK-iT Alexa Fluor red fluorescent oligo, 465318; Invitrogen), Dnmt1 or Pak6 siRNA (Pak6 siRNA sc44879; Dnmt1 siRNA sc-35203; Santa Cruz Biotechnology) for 5 h in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific).

Techniques: Expressing

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

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 1. 5hmC content is diluted during replication. A. Hemi-hydroxymethylated DNA (CG/5hmCG) is not a good substrate for Dnmt1. The DNA methylation activity of mouse Dnmt1, Dnmt3a, and Dnmt3b towards 35-bp unmethylated (CG/CG), hemi- methylated (CG/5mCG), or hemi-hydroxymethylated (CG/5hmCG) DNA was determined. B. Gel mobility shift assaying of the SRA domain of mouse Uhrf1. The indicated concentrations of SRA were incubated with either 12-bp CG/5mC, CG/5hmCG, or CG/CG, followed by electrophoresis (left panel). The complex of the SRA and 32P-labeled CG/5mCG was competed with the indicated amounts of non-labeled CG/5mCG, CG/5hmCG, or CG/CG DNA (right panel). DNA bound to SRA (B) and free DNA (F) are indicated.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: DNA Methylation Assay, Activity Assay, Methylation, Mobility Shift, Incubation, Electrophoresis, Labeling

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 3. Dnmt3a and Dnmt3b mainly provide 5mC for the hydroxymethylation in mESCs. The 5mC, 5hmC, and Tet mRNA contents of J1 parent, Dnmt1 (1-KO), Dnmt3a and Dnmt3b (3-DKO), Dnmt3a (3a-KO), Dnmt3b (3b-KO), and Dnmt1, Dnmt3a and Dnmt3b (TKO) knockout mESCs, and ectopically expressed TAP-tagged Dnmt3a (3a-TAP) or Dnmt3a2 (3a2-TAP) in 3-DKO mESCs were determined. A. The 5mC contents (%) were determined as M.SssI methylation ability from a standard curve (Figure S1A). B. The 5hmC contents were determined from the standard curve obtained on β-GT assaying (Figure S1B). The values are the averages ± SD determined for three independent genomic DNA samples. C. Relative mRNA expression of Tet1, Tet2, and Tet3 was evaluated by semi-quantitative RT-PCR. (-) indicates the product of PCR without the template.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: Knock-Out, Methylation, Expressing, Quantitative RT-PCR

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 5. Dnmt3a and Dnmt3b-dependent 5mC are responsible for the production of 5hmC. The 5hmC (A) and 5mC (B) contents of J1 (blue bars), Dnmt1 (1-KO, red bars), and Dnmt3a and Dnmt3b (3-DKO, light green bars) knockout mESCs were determined by q-PCR in the promoters of five representative 5hmC-enriched genes. The values are the averages + SD determined for three independent genomic DNA samples.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: Knock-Out

Journal: PloS one

Article Title: Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

doi: 10.1371/journal.pone.0082961

Figure Lengend Snippet: Figure 6. Dnmt1, Dnmt3a, Dnmt3b, and Tet1 are recruited to 5hmC-enriched promoters. The occupancy of Dnmt1, Dnmt3a, and Dnmt3b (A), and Tet1, Tet2, and Tet3 (B) was determined by ChIP-qPCR in the promoters of the 5hmC- enriched genes shown in Figure 4. The values are the averages + SD determined for three independent DNA samples.

Article Snippet: Solubilized chromatin was incubated with mouse monoclonal IgG (Cat No. 12-371, Millipore), rabbit IgG (Cat No. 12-370, Millipore), anti-Dnmt1 mouse monoclonal Dnmt1 (clone: 60B1220.1, Cat No. IMG-261A, Imgenex), anti-Dnmt3a/3a2 [5], anti-Dnmt3b [2], anti-Tet1 (Cat. No. 09-872, Millipore), antiTet2 (Cat No. R1086-6b, Abiocode), or anti-Tet3 (Cat. No. 61395, Activemotif) antibodies at 4°C overnight.

Techniques: ChIP-qPCR