Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2 and TET3 associate with the O -GlcNAc transferase OGT and promote GlcNAcylation. ( A ) Silver stain gel of HaloTag-TET protein complex isolations and HaloTag alone control (Ctrl). Protein pulldowns were performed from HEK293T cells overexpressing the indicated HT constructs (see Materials and methods and for details). As not all of the indicated complex isolations were performed at the same time, two separate silver stain gels were run, as shown in this panel. ( B ) Table of transcriptional or chromatin protein interactors found in the various HaloTag-TET isolations. Spectral counts for each interacting protein are shown for biological replicates. TET1, but not TET2, as previously reported ( ; ), shows interaction with SIN3A. OGT interacts with all TET proteins, though it is most highly abundant with TET2 and TET3. ( C ) Detection of OGT by western blotting from HT-TET2 and HT-TET3 pulldowns from ( A ). The indicated pulldowns were probed with an anti-OGT antibody to detect the presence of OGT. OGT and beta-Actin shown as input loading controls. ( D ) TET2 and TET3 co-immunoprecipitate (CoIP) with endogenous OGT from untransfected HEK293T cells. Cell extracts were immunoprecipitated with anti-OGT or rabbit IgG and probed with antibodies against the indicated proteins. An IP control of OGT alone is shown to demonstrate specific capture and enrichment of OGT. Inputs loading controls are shown for all. Note that in this experiment very weak expression of TET2 relative to TET3 is observed. ( E ) The global level of hmC does not change after cell treatment with Alloxan or PUGNAc. Dot blot quantification of global hmC after the indicated treatments. The hmC content is normalized with respect to the input DNA and to mock-treated cells, where the ratio is set at 1.00. Error bars indicate s.d. of three independent experiments. As controls, western blots using anti- O -GlcNAc antibody show the expected decrease in GlcNAcylation with Alloxan and increase with PUGNAc. HDAC1 input loading controls are also shown. Vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( F ) Global decrease in GlcNAcylation is observed in TET2/3 knockdowns. Left: TET2 kd or TET3 kd show decreased GlcNAc activity. Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl, RNAi TET2, or RNAi TET3, and UDP-[ 3 H]GlcNAc incorporation was measured. The amount incorporated into the control cells was set at 1. Error bars indicate s.d. of three independent experiments (* P <0.05). Right: Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl or RNAi TET2/3 and global GlcNAcylation was visualized with an antibody against O -GlcNAc. HDAC1 input loading control is also shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Silver Staining, Control, Construct, Western Blot, Immunoprecipitation, Expressing, Dot Blot, Activity Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3–OGT show genomic co-localization around TSSs and impact on H3K4me3 and transcriptional activation. ( A ) Left: Venn diagrams indicating significant overlap of TET2 and OGT bound regions (left part; P -value<10 −10 ) identified after HaloCHIP-Seq in HEK293T cells expressing HT-TET2, or HT-OGT. Right: TET2–OGT targets are primarily found at TSSs and CpG-rich sequences. Similar profiles were also observed for TET3–OGT . ( B ) An analysed subset of TET2–TET3–OGT targets show a lack of DNA methylation and hydroxymethylation, yet display GlcNAcylation. qPCR analysis of TET2–TET3–OGT binding and non-binding regions after MeDIP (top), hMeDIP (middle), or ChIP with an anti- O -GlcNAc antibody (bottom). ‘% Input' represents real-time qPCR values normalized with respect to the input chromatin. Known methylated and hydroxymethylated regions are shown as positive controls in MeDIP and hMeDIP panels.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Binding Assay, Methylated DNA Immunoprecipitation, Methylation

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: ( C ) TET2/3–OGT targets in HEK293T cells are enriched for H3K4me3 as depicted in a Venn diagram; P -value<10 −10 . ( D ) Examples of HaloCHIP-Seq OGT, TET2, TET3, and ChIP-Seq H3K4me3 profiles (UCSC tracks). ( E ) Decreased levels of H3K4me3 in TET2 kd cells. Upper-left: decrease in the normalized number of H3K4me3 reads in TET2/3–OGT-binding regions in TET2 kd cells versus control RNAi-treated cells. Upper-right: pie chart showing the percentage of TET2–TET3–OGT binding regions with a statistically significant reduction of the normalized number of H3K4me3 reads for TET2 kd versus control RNAi-treated cells. Lower-part: examples of H3K4me3 ChIP-Seq profiles (UCSC tracks) in TET2–TET3–OGT-binding regions for the RNAi control versus TET2 kd sample. ( F ) Western blot showing global decrease in H3K4me3 in a TET2/3 double kd cells. Lysates from mock HEK293T RNAi kd or TET2/3 kd cells were probed for H3K4me3 using an anti-H3K4 antibody in western blot. Tubulin is shown as a loading control. ( G ) OGT activity is important for H3K4me3. Cell extracts were prepared from HEK293T cells treated with or without the OGT inhibitor Alloxan, and then western blots for H3K4me3 were performed. HDAC1 and H3 are shown as loading controls and a western blot against O -GlcNAc was used to monitor specific GlcNAcylation inhibition by Alloxan. Vertical lines indicate juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( H ) Decreases in transcription are observed in both TET2/3 knockdowns and an OGT knockdown. The indicated target genes (which showed decrease in H3K4me3 after TET2 kd; cf. E ) and negative controls (unbound TET2/3–OGT–H3K4me3 targets), were analysed by RT–qPCR in HEK293T cells subjected to the various listed RNAi treatments. Independent experiments were performed in duplicates.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Control, Western Blot, Activity Assay, Inhibition, Knockdown, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin. ( A ) Mass spectrometry reveals HCF1, a known target of OGT and component of SET1/COMPASS , as an interacting partner of HT-TET2 and HT-TET3. Biological duplicates and respective spectral counts (SpC) for HCF1 are shown. ( B ) Protein pulldowns of HT-OGT coupled with mass spectrometry identify HCF1, TET2, TET3, and all components of SET1/COMPASS as partners of OGT. Biological duplicates and SpC for each protein identified are shown for HT-OGT and Ctrl isolations. ( C ) The interaction of HCF1 and SET1/COMPASS components with HT-OGT depends on O -GlcNAc activity. Plot showing average SpCs for HCF1 and SET1/COMPASS components isolated from HT-OGT pulldowns of untreated (grey bars) and Alloxan-treated (green bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( D ) The interaction of HT-SETD1A with SET1/COMPASS components and OGT is reduced by a TET2/3 double kd. Plot showing average SpCs for SET1/COMPASS components and OGT isolated from HT-SETD1A pulldowns of control RNAi-treated (grey bars) and TET2/3 kd (blue bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( E ) A significant reduction in HCF1 GlcNAcylation is observed after TET2/3 double kd. Upper diagram shows a schematic representation of full-length HCF1 and its domains . The GlcNAcylated peptides identified by mass spectrometry from HT-SETD1A isolations from control RNAi-treated and TET2/3 kd cells are indicated below. The full-length HCF1 amino-acid sequence (NP_005325.2) shows the corresponding GlcNAcylated peptides highlighted in yellow with RNAi Ctrl on the left and RNAi TET2/3 kd on the right. ( F ) Bioluminescence resonance energy transfer (BRET) assays show reduction of SETD1A binding to histone H3.3 in the presence of an OGT inhibitor and in TET2/3 kd cells. Upper diagram showing the schematic of BRET energy transfer upon binding of a NanoLuc-SETD1A fusion donor and fluorescently labelled Histone H3.3-HaloTag fusion acceptor in live HEK293T cells (see Materials and methods for experimental details and calculation of BRET). Left: BRET measurements were calculated without treatment (grey) or with Alloxan treatment (green). Right: BRET measurement for RNAi control (grey) or RNAi TET2/3 (blue). Biological triplicates ±s.d. are shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Mass Spectrometry, Isolation, Control, Sequencing, Bioluminescence Resonance Energy Transfer

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters. ( A ) Genome-wide co-localization of endogenous Tet2 with O -GlcNAc and H3K4me3 at promoters and CpG-rich regions. Venn diagrams are shown ( P -value overlap<10 −10 ) as well as the indicated genome-wide distribution. ( B ) Tet2, O -GlcNAc, and H3K4me3 are enriched at many active genes, mirroring the presence of RNA Pol II. Upper panel: Venn diagram showing the overlap of Tet2 and O -GlcNAc with RNA Pol II ( P -value overlap <10 −10 ); lower panel: Box plots showing the reads density at targets and non-targets (others) for ChIP-Seq RNA Pol II or RNA-Seq in mouse bone marrow. ( C ) Global decrease in GlcNAcylation is observed in Tet2 knockout mouse bone marrow. Mouse bone marrow tissues with or without a Tet2 knockout were analysed by western blot for O -GlcNAc levels using an anti- O -GlcNAc antibody. HDAC1 is shown as loading control. ( D ) ChIP-Seq for H3K4me3 in Tet2 knockout mouse tissues shows reduced global H3K4me3 at target promoters. Overall impact on H3K4me3 peak significance (−log( P -value) of the peaks) between wild-type and Tet2 knockout bone marrow is shown. ( E ) Table showing key haematopoietic genes with specifically reduced H3K4me3 in Tet2 knockout as compared to wild type. The location at CpG islands and the promoter class for each is listed. Lower part: example of H3K4me3 ChIP-Seq profiles (UCSC tracks) in wild type versus Tet2 knockout.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Knock-Out, Genome Wide, ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Model connecting DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. Based on our findings, a hierarchical model of the involved proteins, with the cascade of their respective activities, can be envisaged as followed: (1) The first sequence of events in the cascade is the formation of TET2/3–OGT interaction, which promotes OGT GlcNAcylation on numerous proteins, including HCF1; (2) In a TET-dependent manner, a GlcNAcylated HCF1 is important for the formation of the SET1/COMPASS; (3) In the last step, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Sequencing, Activity Assay, Binding Assay, Activation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: Interactome of MALAT1 lncRNA by RAT-seq. A. RAT-seq assay. MALAT1 lncRNA was in situ reverse transcribed using MALAT1-specific complementary primers at 60°C with biotin-dCTP. The biotin-MALAT1 cDNA chromatin complex was isolated by streptavidin beads and cDNAs were isolated for Illumina library sequencing. RAT-seq will generate a genome-wide target interaction network for MALAT1 lncRNA in breast cancer cells. B. Gene ontology enrichment pathway analysis of the MALAT1 RAT-Seq data. GO enrichment was analyzed with Cytoscape software. C. The MALAT1 RAT-seq interactome. The MALAT1 interactome was drawn based on the enrichment fold of the top RAT-Seq pathway target genes.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: In Situ, Reverse Transcription, Isolation, Sequencing, Genome Wide, Software

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 binds to the EEF1A1 promoter and epigenetically regulates its activity. A. The RAT-seq IGV binding of MALAT1 lncRNA at the EEF1A1 locus. MALAT1-RAT: the RAT-seq library created by the MALAT1-specific complementary primers; RC-RAT: the RAT-seq control library created by random oligonucleotide primers; pEEF1A1: EEF1A1 promoter; 3’-CT, 5’-CT: the 3’- and 5’-control sites; E1-E8: EEF1A1 exons. B. Quantitation of EEF1A1 binding in the MALAT1-specific RAT-seq products and the negative control RAT-seq products. C. EEF1A1 expression levels by Q-PCR in MALAT1-knockdown cells. β-Actin was used as an internal control. **P < 0.01 as compared with the control groups. D. Western blot of eEF1A1. Note the reduced expression of eEF1A1 in MALAT1-knockdown breast cancer cells. GAPDH was used as control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Activity Assay, Binding Assay, Control, Quantitation Assay, Negative Control, Expressing, Knockdown, Western Blot

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 epigenetically regulates EF1A1. A. pEEF1A1-luciferase assay. The EEF1A1 promoter (pEEF1A1) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. **P < 0.01 as compared with the control groups. B. Quantitation of Histone 3-K4 (H3K4) trimethylation. All data are presented as the relative values after normalization over the input DNA. *P < 0.05 as compared with control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Luciferase, Sequencing, Clone Assay, Reporter Assay, Plasmid Preparation, Negative Control, Control, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 is dysregulated in breast cancer. A. Unsupervised hierarchical clustering analysis of the significantly differentially expressed genes in paracancer tissues and tumor tissues. Data from 64 mammary tissues was downloaded from TCGA database. In the heatmap, the normalized expression values are represented in shades of green and red, indicating the expression being above and below the median expression value across the samples. B. The normalized expression level of MALAT1 in 64 mammary tissues (32 paracancer tissues and 32 tumor tissues). ***P < 0.01 as compared with the paracancer group. C. Q-PCR quantitation of MALAT1 expression in breast cancer cell lines.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Expressing, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: The role of MALAT1 in cell proliferation and cell cycle. A. MALAT1 shRNA knockdown in two breast cancer cell lines. MALAT1 expression was examined by Q-PCR in CTL (non-transfected control), shNC (random shRNA non-targeting control), shMALAT1 (MALAT1 shRNA-1 transfected cells), and shMALAT1-2 (MALAT1 shRNA-2 transfected cells). β-Actin was used as an internal control. **P < 0.01 as compared with CTL and shNC controls. B. Cell Proliferation. CCK-8 assay was used to determine cell growth viability at 0, 24, 48, 72 and 96 hour time points. C. Cell cycle. Flow cytometry was used to measure cell cycle profile with propidium iodide staining. Cell numbers were counted according to DNA content of G0/G1, S and G2/M phases. The statistical results are shown on the right panel. *P < 0.05 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, shRNA, Control, Transfection, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: EEF1A1 rescues the effect induced by MALAT1 knockdown. A. Overexpression of EEF1A1 in breast cancer cells. The expression of EEF1A1 was quantitated by Q-PCR. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. β-Actin was used as an internal control. ***P < 0.001 as compared with the control groups. Overexpression of eEF1A1 in breast cancer cells was measured by Western blot. B. Cell growth viability as measured by CCK-8 assay. C. Cell invasion as examined by Transwell assay. Quantitation of invaded cells was shown as mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, Over Expression, Expressing, shRNA, Control, Transfection, Western Blot, CCK-8 Assay, Transwell Assay, Quantitation Assay

Journal: Journal of biomedical science

Article Title: SLCO4A1-AS1 promotes colorectal tumourigenesis by regulating Cdk2/c-Myc signalling.

doi: 10.1186/s12929-022-00789-z

Figure Lengend Snippet: Fig. 2 Promoter hypomethylation promotes SLCO4A1-AS1 expression in CRC. a Schematic map illustrating a predicted CpG island and its DNA methylation probes in the promoter of SLCO4A1-AS1. TSS, transcription start site. b The β-value of methylation of SLCO4A1-AS1 was lower in tumour samples than in normal tissues according to the CRC dataset of TCGA. The β-value of methylation of SLCO4A1-AS1 was linearly related to SLCO4A1-AS1 expression in CRC tissues from TCGA (c) and cell lines from CCLE (d). e Relative expression of SLCO4A1-AS1 in CRC cell lines was measured using qRT-PCR (the left panel). The methylation levels of SLCO4A1-AS1 in CRC cell lines and leukocyte cells were determined by bisulfite sequencing PCR. A total of 5 individual clones were randomly picked for sequencing (the right panel). f The mean methylation levels of these CpG sites were negatively associated with the expression levels of SLCO4A1-AS1 in CRC cells. g SLCO4A1-AS1 expression in CRC cells treated with DNA methyltransferase inhibitor (5-aza-dC). h DNA methylation analyses of SLCO4A1-AS1 in paired CRC tissues and noncancerous tissues using methylation-specific PCR assay. N, adjacent noncancerous tissue; T, tumour tissue; M, DNA marker

Article Snippet: Genomic DNA was extracted from cancer cells or human leukocytes using a Genomic DNA Mini Preparation Kit (Beyotime, China) and then bisulfite-modified using an EpiJET Bisulfite Conversion Kit (Thermo Fisher, USA).

Techniques: Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, Sequencing, Marker

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Schematic outline of alk-BER assay. The assay involves exposing the cells to MMS (step 1), isolation of total genomic DNA (step 2), conversion of MMS-induced methylated bases to SSBs with damage specific enzymes AAG and APE1 (step 3), separation of DNA fragments containing SSBs by alkaline agarose gel electrophoresis (step 4), gel staining, imaging, and quantitation of MDAs (step 5).

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Isolation, Methylation, Agarose Gel Electrophoresis, Staining, Imaging, Quantitation Assay

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Alk-BER assay in yeast cells ( S. cerevisiae ). ( A ) Representative alkaline agarose gel image of MMS-induced DNA damage dose response in the BY4741 strain of S. cerevisiae . Genomic DNA of cells not exposed to MMS (C: control), and DNA of cells exposed to increasing doses of MMS (5, 10, or 20 mM) was resolved on alkaline agarose gel. Each DNA sample was treated with (+) and without (−) a cocktail of AAG and APE1 enzymes. ( B ) Dose dependent increase in the numbers of MMS-induced methyl G, A per 1 kb DNA fragment. Each data point denotes the average value and standard deviation of three independent experiments. ( C ) Representative gel image of DNA damage and repair time course in the BY4741 strain of S. cerevisiae . M: DNA size standard lambda/HindIII. C: control, cells not exposed to MMS, 0: cells collected after 10 min exposure to 20 mM MMS, 1–3 h: cells collected after 1, 2, 3 h of repair. ( D ) Quantitative representation of data displayed in panel C. Formation and repair of MMS-induced methyl G and A (7meG, 3meA), as a function of repair time. Each data point represents an average of 3 independent experiments; error bars were calculated based on standard deviation. Gel image presented in panel ( A ) has been cropped. Original, uncropped gel image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Agarose Gel Electrophoresis, Standard Deviation

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: Alk-BER assay in human cells. ( A ) MMS dose response in SW13 cells. Cells were treated with increasing doses of MMS for 10 min at RT. Representative alkaline agarose gel image is shown. ( B ) Quantification of methyl A, G per 1 kb DNA fragment as a function of increasing MMS dose. The graph represents quantification of the data in panel ( A ). ( C ) Efficiency of double enzyme (AAG&APE1), and single enzymes: (AAG only), and (APE1 only), in converting methyl DNA adducts to SSBs. ( D ) Alkaline gel image representing DNA damage and repair time course. SW13 cells were exposed to 10 mM MMS for 10 min. Genomic DNA was isolated, and processed with double enzyme AAG&APE1 digest. ( E ) Quantification of the repair and removal of methyl A,G as a function of time. ( F ) SW13 cell viability measured by trypan blue. Each data point represents an average of 3 independent experiments; error bars were calculated based on standard deviation. Gel image presented in panel ( C ) has been cropped. Original, uncropped gel image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Agarose Gel Electrophoresis, Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Versatile cell-based assay for measuring DNA alkylation damage and its repair

doi: 10.1038/s41598-021-97523-w

Figure Lengend Snippet: The repair of MDAs is slow in human cells and does not correlate well with the levels of endogenous AAG enzyme. DNA damage and repair time course experiment was performed in several human cell lines; CHON-002, SW13, and HAP1. Cells (60–70% confluent) were exposed to 10 mM (0.1%) MMS in 1xPBS for 10 min at RT, followed by DNA repair time course 0, 3, 8 and 22 h at 37 °C. ( A ) DNA repair rates were quantitated individually for each cell line and expressed as a percentage (%) of the removed methyl A,G, as compared to the 0 h time point. Each data point represents an average of two independent experiments. ( B ) Endogenous levels of AAG enzyme were detected by Western blotting. Western blot image presented in panel ( B ) has been cropped. Original, uncropped blot image is included in the supplementary data.

Article Snippet: Next, fresh pre-warmed media were added and cells were allowed to repair for 0, 3, 8, or 22 h. GM 12878 cells were treated with 5 mM (0.05%) MMS for 5 min. Total genomic DNA was purified from each time point using the PureLink genomic DNA mini kit (K 182001, Thermo Fisher Scientific).

Techniques: Western Blot