Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Schematic diagram of the positions and orientations of syncytin-1, PEX1 and GATAD1 genes. Arrows show genes’ orientations. The patterned squares represent exons. Dark squares indicate the location of the CpG islands in GATAD1 gene. Light grey square represents the syncytin-1 5′ LTR region. The solid lines at the bottom show the positions of amplicons of real-time PCR. Note the opposite orientations of GATAD1 and syncytin-1 genes, which bring the 3 [prime] region of GATAD1 to a closer vicinity of syncytin-1 gene.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Information of PCR primers

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques:

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: PEX1 and GATAD1 mRNA levels in first-trimester (1N, n=8), third-trimester normal (3N, n=14) and third-trimester preeclamptic (3P, =7) placentas. Real-time PCR were performed as described in Materials and methods. Data were standardized by the results from β-actin internal control. The mRNA levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors of each group were presented. a PEX1 mRNA levels. No significant difference was found between 1N and 3P, or 3N and 3P groups. b GATAD1 mRNA levels. Significantly higher GATAD1 mRNA levels were observed in 3N than in 1N; A significant reduction of GATAD1 mRNA levels were found in 3P compared to 3N group. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Protein levels of GATAD1 in the first-trimester (1N1-1N6), third-trimester normal (3N1-3N6), and third-trimester preeclamptic (3P1-3P6) placentas. The sizes of GATAD1 and β-actin proteins are 29 kDa and 42 kDa, respectively. a Western blotting performed using GATAD1-specifc antibodies. b Results of densitometry analyses showing a similar trend of changes to that of mRNA levels: GATAD1 protein expression was higher in 3N than 1N, and lower in 3P than 3N. The GATAD1 expression data were standardized by the results from β-actin. The protein levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors from each group were presented. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Representative results of immunohistochemistry (40×10). The paraffin-embedded placental tissues were sliced into 4 μm sections. The sections were processed as describe under Materials and methods. As negative control (bottom right panel), a section of first-trimester normal placenta was processed with the same procedures except for the absence of primary antibodies. GATAD1 protein was stained brown color. The nuclei were stained blue with haematoxylin. GATAD1 protein localized mostly in the cytoplasm and membrane of syncytiotrophoblasts (ST), and to a less extent, the cytoplasm and membrane of cytotrophoblasts (CT). Higher level of GATAD1 expression was found in third-trimester (upper right) than first-trimester (upper left panel) placenta. Preeclamptic (bottom left) placentas expressed decreased levels of GATAD1 protein compared to normal placentas.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Negative Control, Staining, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: GATAD1 gene methylation measured by COBRA. Following PCR amplification, DNA fragments representing the 5 [prime] and 3 [prime] regions of GATAD1 gene were digested with an excess of BstUI or TaqαI, respectively. Agarose gel electrophoresis was performed and DNA bands were visualized by ethidium bromide staining. a The absence of cleavage product (supposedly 147 bp and 107 bp) from 254 bp fragment indicated an largely unmethylated status of GATAD1 5 [prime] region in first-trimester (1N1 to 1N8), third-trimester normal (3N1 to 3N14) and Preeclamptic (3P1 to 3P7) placentas. b The 241 bp fragment representing the 3 [prime] region of GATAD1 was mostly cleaved, generating the 144 bp and 97 bp bands indicative of DNA methylation. c Densitometry analyses of the 3 [prime] methylation showing an increased methylation in 3N placentas compared to 1N, and decreased methylation levels in 3P placentas compared to 3N group. ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation, Combined Bisulfite Restriction Analysis Assay, Amplification, Agarose Gel Electrophoresis, Staining, DNA Methylation Assay

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Bisulfite sequencing of the GATAD1 3 [prime] region. Bisulfite-converted DNA from 1N (n=5), 3N (n=5) and 3P (n=5) groups were PCR amplified, subcloned, and sequenced. a The typical sequencing result of the 3 [prime] region. Asterisks (*) mark CpG sites. The TaqαI recognition site used in COBRA is underlined. b GATAD1 3 [prime] bisulfate sequencing results. The solid and open circles represent the methylated and unmethylated cytosines, respectively, in CpGs dinucleotides contexts. The average methylation levels for each CpG site were presented in the bottom panels. c Quantitative comparison of the GATAD1 3 [prime] methylation among the three groups. 3N placentas displayed increased methylation levels compared to 1N, and 3P group exhibited decreased methylation levels compared to 3N. * p < 0.05; ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation Sequencing, Amplification, Sequencing, Combined Bisulfite Restriction Analysis Assay, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: The correlation between GATAD1 expression and GATAD1 3 [prime] methylation in human placentas (n=29). The Y-axis indicated GATAD1 mRNA levels and the X-axis represented GATAD1 3 [prime] methylation index. Spearman correlation analysis showed a highly significant positive correlation between GATAD1 mRNA levels and GATAD1 3 [prime] methylation levels among placental samples (r=0.62, p=0.0003).

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Expressing, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Treatment with DNMT inhibitor led to a decreased GATAD1 3 [prime] DNA methylation and decreased GATAD1 expression. JAR cells were treated for 5 days with 0, 0.5, and 2.5 μM of 5-aza-deoxycytidine (ADC). a GATAD1 3 [prime] methylation was examined with COBRA. b Densitometry analyses indicated a dose-dependent decrease of GATAD1 3 [prime] DNA methylation following ADC treatment. c Results of real-time PCR showed a decrease of GATAD1 mRNA expression following ADC treatment. Data were standardized with the results from β-actin. Averages and standard errors were presented in the chart. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: DNA Methylation Assay, Expressing, Methylation, Combined Bisulfite Restriction Analysis Assay, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 1. High glucose increases O-GlcNAcylation of DNMT1 in cell lines and primary cells. (A) Hep3B cells were treated with glucose (5 mM or 25 mM) with or without Thiamet-G (TMG). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). (B) Lysates of Hep3B treated with glucose were immunoprecipitated with DNMT1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc (n = 3). (C) Peripheral blood mononuclear cells (PBMCs) were isolated from three individual donor blood samples and treated with increasing concentration of glucose for 24 hr. Collected cell lysates from PBMCs were immunoprecipitated with antibody targeting DNMT1 and immunoblotted for O-GlcNAc. Representative blot from one donor (n = 3). (D) Immunoblots for O-GlcNAc and GAPDH from liver samples of C57BL/6J mice given a high- fat/high-sucrose diet (HF/HS) or normal diet (chow) for 4 mo, and immunoprecipitated with Dnmt1. Lysates of mouse liver were immunoprecipitated with Dnmt1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc. *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A-D); ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Western Blot, Immunoprecipitation, Isolation, Concentration Assay

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 2. Identification of O-GlcNAcylated sites within DNMT1 by LC-MS/MS. (A) Schematic drawing of the DNMT1 O-GlcNAc-modified region enriched from Hep3B cells based on mass spectrometry (MS) data and tandem MS (MS/MS) peaks. FTMS+ p NSI full MS (400.0000–1600.0000). DQDYARFESPPKTQPTEDNKF (S9 HexNAc) – S878. (B) Schematic diagram of identified novel O-GlcNAcylated and phosphorylated sites within DNMT1 as determined via LC-MS/MS. DMAP, DNA methyltransferase associated protein-binding domain; PCNA, proliferating cell nuclear antigen-binding domain; NLS, nuclear localization sequences; RFTS, replication foci targeting sequence domain; BAH, bromo-adjacent homology domain. (C) Sequence conservation of S878 in vertebrates. (D) Each immunoprecipitated Myc-DNMT1 wild type and substituted mutants was immunoblotted with an O- GlcNAc antibody (n = 3). **p<0.0005; ***p<0.0001 by Student’s t-test (D); N.D., not detected, ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Liquid Chromatography with Mass Spectroscopy, Modification, Mass Spectrometry, Tandem Mass Spectroscopy, Protein Binding, Binding Assay, Sequencing, Immunoprecipitation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 4. High glucose leads to loss of DNA methylation at cancer-specific partially methylated domains (PMDs). (A) Density plot of DNA methylation for DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Genome browser screenshot of DNA methylation for DNMT1-WT and DNMT1-S878A cells and low or high glucose along with liver tumor PMDs from Li et al., 2016. (C) Boxplots of DNA methylation at PMDs or general genomic background (BG) for each DNMT1-WT and DNMT1-S878A treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). (D) Heatmap representation of global DNA methylation for DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc) at gene-poor and gene-rich regions. (E) Methylation changes from O-GlcNAcylation of DNMT1 by wave score for replication timing (Hansen et al., 2010; Thurman et al., 2007). ***p<0.0001 by Wilcoxon signed-rank test (C).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: DNA Methylation Assay, Methylation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 5. High glucose-induced reactive oxygen species (ROS) and DNA damage cause apoptotic cell death in DNMT1-WT cells. (A) Quantitative fluorescence image of ROS in DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Quantitative fluorescence image of γ-H2A.X in DNMT1-WT and DNMT1-S878A cells treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O- GlcNAc). (C) Quantitative fluorescence image of cell death in propidium iodide staining of DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A–C); data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Fluorescence, Staining