Journal: Cancers

Article Title: Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation

doi: 10.3390/cancers12020447

Figure Lengend Snippet: ( A ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to the indicated concentrations of 2b or 4c . Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as loading control. Blots are representative of two independent experiments. Right: Densitometric analysis of protein levels is reported. ( B ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to 4c at 1 µM and co-treated with bortezomib (when indicated) used at 10 nM. Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as a loading control. Blots are representative of three independent experiments. Right: Densitometric analysis of protein levels is reported. Data are represented as mean ± SEM. Significance is represented as * p < 0.05 related to the control.

Article Snippet: The following primary antibodies were used for immunoblotting: α-DNMT1 (Novus Biologicals, Littleton, CO, USA). α-DNMT3a (SantaCruz Bio-technologies, CA, USA) and α-GAPDH (Millipore Corp., Bedford, MA USA), used as a loading control.

Techniques: Western Blot, Expressing, Control

Journal:

Article Title: Characterization of Dnmt3b:thymine-DNA glycosylase interaction and stimulation of thymine glycosylase-mediated repair by DNA methyltransferase(s) and RNA

doi: 10.1016/j.jmb.2008.02.049

Figure Lengend Snippet: DNA methyltransferases stimulate base excision repair of T·G mismatches. A: Design and testing of oligodeoxyribonucleotides (ODNs) used in a mismatch repair assay. (a) Sequence of the double-stranded ODN. The sense strand contains either a normal CCGG site or a mutated CTGG site. The antisense strand contains either C or 5mC within an MspI/HpaII recognition site. Positions of variable nucleotides y and x are shown in bold. The MspI/HpaII recognition sequence is indicated with a bar. The asterisk indicates the position of the 32P label. (b) Predicted sensitivity to MspI or HpaII digestion of the control C·GC and C·GmC ODNs compared to the mismatched T·GC and T·GmC ODNs. (c) MspI digestion of the control ODNs results in the expected 26 nt long radiolabeled fragment from both controls, while a 26 nt long fragment is only observed after HpaII digestion of the unmethylated control. In contrast, T·G mismatch ODNs are refractory to digestion by either enzyme. M, MspI. H, HpaII. B: De novo DNA methyltrasnferases stimulate repair of T·G mismatches. (a) Evaluation of Dnmt and Tdg protein levels in J1 and Dnmt null ES cells. Nuclear extracts prepared from each cell line were immunoblotted using antibodies specific for Dnmt1, Dnmt3a, Dnmt3b and Tdg to verify the expression of Tdg and the absence of specific Dnmt expression in each cell line relative to wild-type J1. Immunoblot for Pcna served as a loading control. (b) Predicted T·GC ODN sensitivity to digestion by MspI or HpaII depending on extent of repair/DNA methylation. (c) Typical results from one of three independent experiments in which the T·GC ODN was incubated with wild-type J1 or Dnmt null ES cell nuclear extracts followed by digestion with either MspI or HpaII. The expected 26 nt repair product produced as a result of digestion is indicated by the arrowhead. In the absence of either Dnmt3b and/or Dnmt3a there is a reduction in repair efficiency when compared to wild-type nuclear extracts (refer to text for discussion of Dnmt1 null extracts). As a control (last lane), T·GC ODN was used in a repair assay but was not digested. Lack of a 26 nt fragment indicates the ODN is being repaired and not merely nicked 5′ to the mismatched thymine. (d) Quantitation of differences in the extent of mismatch repair by extracts from normal ES cells (J1) and ES cells nullizygous for the indicated Dnmts. Error bars represent the mean (± SD) of three independent experiments. [*] P < 0.005

Article Snippet: Protein was transferred to PVDF membrane and analyzed by standard immunoblotting with protein specific antibodies: α-Dnmt1 (Santa Cruz, K-18), α-Dnmt3a (Imgenex), α-Dnmt3b (Imgenex), α-Tdg( 37 ), α-PCNA (Santa Cruz, sc-56) with appropriate secondary antibodies conjugated to horse radish peroxidase (HRP).

Techniques: Sequencing, Expressing, Western Blot, DNA Methylation Assay, Incubation, Produced, Quantitation Assay

Journal: PLoS ONE

Article Title: A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines

doi: 10.1371/journal.pone.0009001

Figure Lengend Snippet: Cells were treated daily with AZA or DAC (0–3 µM in KG-1a; 0–10 µM in THP-1) for 48 and 72 hours. Protein lysates were analyzed by Western analysis for DNMT1 and phospho-H2AX (Ser 139) proteins. α-Tubulin is shown as a protein loading control. AZA = azacitidine; DAC = decitabine; DNMT = DNA methyltransferase.

Article Snippet: The α-tubulin and DNMT1 antibodies were from EMD Chemicals Inc. (Gibbstown, NJ) and Abcam Inc. (Cambridge, MA), respectively.

Techniques: Western Blot, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: MicroRNA dependent regulation of DNMT-1 and tumor suppressor gene expression by Interleukin-6 in human malignant cholangiocytes

doi: 10.1002/hep.23381

Figure Lengend Snippet: Cell lysates were obtained from Mz-ChA-1 or KMCH cells stably transfected to over-express IL-6 or their respective controls. Immunoblot analysis was performed to assess DNMT-1, p16INK4a, and Rassf1a protein expression. The blots were stripped and reprobed for α-tubulin as a loading control and for quantitation. Representative immunoblots are shown along with quantitative data that show the mean ± standard error from 4 separate blots. * p < 0.05 when compared with respective controls.

Article Snippet: Antibodies against DNMT-1 and p16INK4a were obtained from Santa Cruz Biotechnology Inc.; Rassf1A from Abcam (Cambridge, MA); and α-tubulin from Sigma (St. Louis, MO).

Techniques: Stable Transfection, Transfection, Western Blot, Expressing, Quantitation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: MicroRNA dependent regulation of DNMT-1 and tumor suppressor gene expression by Interleukin-6 in human malignant cholangiocytes

doi: 10.1002/hep.23381

Figure Lengend Snippet: Panel A: Mz-ChA-1 cells were transfected with either miR-148a, miR-152, miR-301 or control precursor. After 24 h, real-time PCR assays were performed using TaqMan Human MicroRNA Assay kit and the expression of miRs was normalized to that of the small nuclear RNA U6B (RNU6B). A significant increase in microRNA expression was observed following transfection with precursor miR-148a and miR-152 compared to control miRNA precursors. The data represent the mean and standard errors from four independent transfections. * p < 0.05 relative to respective controls. Panel B: Mz-ChA-1 and KMCH cells were transfected with either miR-148a, miR-152, miR-301 or control precursor. After 72 hours, cell lysates were obtained for Western blot analysis of DNMTs, Rassf1a, and p16INK4a. The blots were stripped and re-probed for α-tubulin as a loading control.

Article Snippet: Antibodies against DNMT-1 and p16INK4a were obtained from Santa Cruz Biotechnology Inc.; Rassf1A from Abcam (Cambridge, MA); and α-tubulin from Sigma (St. Louis, MO).

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

Journal: Hepatology (Baltimore, Md.)

Article Title: MicroRNA dependent regulation of DNMT-1 and tumor suppressor gene expression by Interleukin-6 in human malignant cholangiocytes

doi: 10.1002/hep.23381

Figure Lengend Snippet: Cell lysates were obtained from H69 non-malignant cholangiocytes and KMCH, CC-LP, TFK and MzChA-1 cholangiocarcinoma cell lines. Immunoblot analysis was performed for DNMT-1 and for the tumor suppressor genes RASSF1a, and p16INK4a using antigen specific antibodies. The blots were stripped and reprobed for α-tubulin as a loading control and for quantitation. Representative immunoblots are shown along with quantitative data showing the mean ± standard error from 4 separate blots. For each cholangiocarcinoma cell line, DNMT expression was significantly increased and Rassf1a and p16INK4a expression significantly decreased compared with non-malignant H69 cells, with p<0.05.

Article Snippet: Antibodies against DNMT-1 and p16INK4a were obtained from Santa Cruz Biotechnology Inc.; Rassf1A from Abcam (Cambridge, MA); and α-tubulin from Sigma (St. Louis, MO).

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: MicroRNA dependent regulation of DNMT-1 and tumor suppressor gene expression by Interleukin-6 in human malignant cholangiocytes

doi: 10.1002/hep.23381

Figure Lengend Snippet: IL-6 over-expressing (Mz-IL-6) or control (Mz-1) cells (3×106) were injected subcutaneously into the flank of athymic BALB/c mice. Panel A: Western blot analysis was performed in tissue lysates from tumor cell xenografts using antibodies to DNMT-1, Rassf1a, p16INK4a, and α-tubulin. A representative blot image is shown on left panel whereas the densitometric analysis is shown on right panel. Panel B: miRNA was extracted from xenograft tissues and miR-148a, miR-152 and miR-301 expression was assessed by quantitative real-time PCR. Potential DNMT-1 targeting miRNA expressions were normalized to expression of RNU6B. The data represent results from three experiments performed in triplicate. *p<0.05 when compared with miRNA expression in control cell xenografts.

Article Snippet: Antibodies against DNMT-1 and p16INK4a were obtained from Santa Cruz Biotechnology Inc.; Rassf1A from Abcam (Cambridge, MA); and α-tubulin from Sigma (St. Louis, MO).

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

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphatidylinositol 3‐Kinase– DNA Methyltransferase 1–miR‐1281–Histone Deacetylase 4 Regulatory Axis Mediates Platelet‐Derived Growth Factor–Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

doi: 10.1161/JAHA.117.007572

Figure Lengend Snippet: Platelet‐derived growth factor (PDGF) BB–phosphatidylinositol 3‐kinase– DNA methyltransferase 1 (DNMT 1) activation cassette mediates PDGFBB repression of miR‐1281. A, Expression of DNMT 1 protein increased along with the increase of PDGFBB use. B, Real‐time quantitative reverse transcription–polymerase chain reaction analysis of small interfering (si)‐ DNMT 1 silencing efficiency in pulmonary artery smooth muscle cells (PASMC s) without and with PDGFBB treatments. C, Transfection of si‐ DNMT 1 recovered PDGFBB repression of miR‐1281 in PASMC s. D, Effects of different pathway inhibitors on DNMT 1 expression were analyzed, and pictilisib was identified to inhibit PDGFBB upregulated mRNA level of DNMT 1. E, Pictilisib inhibited PDGFBB upregulation of DNMT 1 in a dose‐dependent manner. F, Pictilisib also inhibited PDGFBB ‐repressed expression of miR‐1281. DMSO indicates dimethyl sulfoxide; NC, negative control; and N.S., not significant. * P <0.05, ** P <0.01, *** P <0.001 compared with control or as specified by broken lines in respective figures. Exact P values in consecutive order are: 0.034235, 0.009507, 0.043504, 0.003449, 0.010124, 0.049599, 0.000249, 0.010004, 0.036281.

Article Snippet: After that, the membrane was incubated with antibodies against α‐smooth muscle actin (Sigma‐Aldrich, Burlington, MA), β‐tubulin and Smooth Muscle (SM)‐22α (Abcam, Cambridge, MA), HDAC4 and DNMT1 (Proteintech, China), and β‐actin and Smoothelin (Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C.

Techniques: Derivative Assay, Activation Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Transfection, Negative Control, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphatidylinositol 3‐Kinase– DNA Methyltransferase 1–miR‐1281–Histone Deacetylase 4 Regulatory Axis Mediates Platelet‐Derived Growth Factor–Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

doi: 10.1161/JAHA.117.007572

Figure Lengend Snippet: Reduced miR‐1281 level is identified in hypoxic pulmonary artery smooth muscle cells ( PASMCs ), rats with pulmonary arterial hypertension (PAH), and patients with PAH. The relative miR‐1281 levels were estimated by real‐time quantitative reverse transcription–polymerase chain reaction in PASMC s exposed to normoxia (21% O 2 ) or hypoxia (3% O 2 ) for 12 and 24 hours (A), pulmonary arteries individually collected from 4 rats with monocrotaline ( MCT )–induced PAH and 4 controls (rats received intraperitoneal injections of normal saline; B), and serum individually collected from 13 healthy participants and 29 patients with coronary heart disease ( CHD )– PAH (C). D, Proposed regulatory model of phosphatidylinositol 3‐kinase (PI3K)– DNA methyltransferase 1 ( DNMT 1)–miR‐1281–histone deacetylase 4 ( HDAC 4) axis. Upward arrow indicates upregulation or activation. Downward arrow indicates downregulation. N.S. indicates not significant; PDGF, platelet‐derived growth factor; and TF , transcription factor. * P <0.05, ** P <0.01 compared with normoxia, saline or normal control. Exact P values in consecutive order are: 0.00332, 0.001079, 0.024024.

Article Snippet: After that, the membrane was incubated with antibodies against α‐smooth muscle actin (Sigma‐Aldrich, Burlington, MA), β‐tubulin and Smooth Muscle (SM)‐22α (Abcam, Cambridge, MA), HDAC4 and DNMT1 (Proteintech, China), and β‐actin and Smoothelin (Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Saline, Histone Deacetylase Assay, Activation Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Extracellular matrix stiffness regulates fibroblast differentiation by influencing DNA methyltransferase 1 expression through microtubule polymerization

doi: 10.1101/2022.10.20.513009

Figure Lengend Snippet: Extracellular matrix stiffness regulates fibroblast-to-myofibroblast transition through DNMT1 (a-c) Western blot and immunohistochemical staining of DNMT1 protein in the anterior vaginal wall of control and POP patients; (d,f) Expression of DNMT1 in cells cultured on gel with different stiffness.(e,f) The protein levels of these genes were detected by Western blot. Cells were treated with 5 ‘-AZA or PBS as control group and cultured on soft PA gel.

Article Snippet: Antibodies used include rabbit polyclonal antibody (pAb) against DNMT1 (purchased from Cell Signaling Technology), rabbit polyclonal antibody against α-SMA, mouse polyclonal antibody against GADPH, rabbit polyclonal antibody against Collagen1, and rabbit polyclonal antibody against Collagen3 antibody (purchased from Proteintech).

Techniques: Western Blot, Immunohistochemical staining, Staining, Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Extracellular matrix stiffness regulates fibroblast differentiation by influencing DNA methyltransferase 1 expression through microtubule polymerization

doi: 10.1101/2022.10.20.513009

Figure Lengend Snippet: Cells convert physical signals to biological signals through the microtubule cytoskeleton(a) Gene correlation analysis showed that DNMT1 expression was significantly correlated with genes related to microtubule polymerization; (b,c) The morphological differences of cells cultured with high stiffness gel and added with Nocodazole were observed under forward microscope.(d,e) The protein levels of these genes were detected by Western blot. Cells were treated with 5 ‘-AZA or PBS as control group and cultured on soft PA gel.

Article Snippet: Antibodies used include rabbit polyclonal antibody (pAb) against DNMT1 (purchased from Cell Signaling Technology), rabbit polyclonal antibody against α-SMA, mouse polyclonal antibody against GADPH, rabbit polyclonal antibody against Collagen1, and rabbit polyclonal antibody against Collagen3 antibody (purchased from Proteintech).

Techniques: Expressing, Cell Culture, Microscopy, Western Blot, Control

Journal: Cancers

Article Title: Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation

doi: 10.3390/cancers12020447

Figure Lengend Snippet: ( A ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to the indicated concentrations of 2b or 4c . Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as loading control. Blots are representative of two independent experiments. Right: Densitometric analysis of protein levels is reported. ( B ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to 4c at 1 µM and co-treated with bortezomib (when indicated) used at 10 nM. Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as a loading control. Blots are representative of three independent experiments. Right: Densitometric analysis of protein levels is reported. Data are represented as mean ± SEM. Significance is represented as * p < 0.05 related to the control.

Article Snippet: The following primary antibodies were used for immunoblotting: α-DNMT1 (Novus Biologicals, Littleton, CO, USA). α-DNMT3a (SantaCruz Bio-technologies, CA, USA) and α-GAPDH (Millipore Corp., Bedford, MA USA), used as a loading control.

Techniques: Western Blot, Expressing, Control