5hmc  (New England Biolabs)


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    Name:
    EpiMark 5 hmC and 5 mC Analysis Kit
    Description:
    EpiMark 5 hmC and 5 mC Analysis Kit 20 rxns
    Catalog Number:
    e3317s
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    258
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    20 rxns
    Category:
    DNA Fragment Analysis Kits
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    Structured Review

    New England Biolabs 5hmc
    EpiMark 5 hmC and 5 mC Analysis Kit
    EpiMark 5 hmC and 5 mC Analysis Kit 20 rxns
    https://www.bioz.com/result/5hmc/product/New England Biolabs
    Average 92 stars, based on 66 article reviews
    Price from $9.99 to $1999.99
    5hmc - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    2) Product Images from "Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation"

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    Journal: Epigenetics

    doi: 10.4161/epi.24280

    Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation
    Figure Legend Snippet: Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation

    Techniques Used: Methylation

    3) Product Images from "α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis"

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0802-8

    CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p
    Figure Legend Snippet: CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Techniques Used: CRISPR, Activity Assay, Western Blot, Plasmid Preparation, Microscopy

    KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p
    Figure Legend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Techniques Used: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

    4) Product Images from "TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation"

    Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.107532

    TET1 is required for the PAX4 enhancer to achieve a hypomethylated state. a Genome-browser view of the PAX4 locus with increased methylation and decreased chromatin associability upon TET depletion at a TET1/FOXA2 co-bound region featuring enhancer signatures H3K4me1 and H3K27ac. b Locus-specific increase in 5mC at the PAX4 enhancer in TKO or TET1KO samples compared with TET2/3DKO samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation.
    Figure Legend Snippet: TET1 is required for the PAX4 enhancer to achieve a hypomethylated state. a Genome-browser view of the PAX4 locus with increased methylation and decreased chromatin associability upon TET depletion at a TET1/FOXA2 co-bound region featuring enhancer signatures H3K4me1 and H3K27ac. b Locus-specific increase in 5mC at the PAX4 enhancer in TKO or TET1KO samples compared with TET2/3DKO samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation.

    Techniques Used: Methylation

    Pancreas-specific hyper-DMRs show reduced chromatin activity during pancreatic differentiation. a Volcano plot of WGBS data illustrating differentially methylated CpGs (DMCs) identified in TKO_PP cells compared with WT_PP cells. Red and blue represent increased and decreased 5mC in TKO_PP cells, respectively (credible methylation difference > 0.2). b Heatmap illustrating methylation difference between TKO_PP and WT_PP cells at centers of annotated genomic features (± 5 kb) for chromatin accessibility (ATAC), hydroxylation (5hmC), TF binding (FOXA2, GATA4, GATA6, and PDX1), bivalent promoters, poised enhancers, and active enhancers. Average 5mC signals of every 100-bp bin were calculated. c Classification of TKO hyper-DMRs based on 5mC levels in hESCs (green), WT_PP cells (blue), and TKO_PP cells (red). d Average plots of FOXA2 (left column), GATA4 (middle column), and GATA6 (right column) signal at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in pancreatic progenitors. e Average plots of ATAC (left column), H3K27ac (middle column), and H3K4me1 (right column) at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in WT_PP (blue) and TKO_PP (red) cells. f Average plots of 5mC at proximal (≤ 1 kb from TSS) and distal ( > 1 kb from TSS) decreased accessible regions in WT_PP (blue) and TKO_PP (red) cells. g Genome-browser view of the PDX1/PDX1-AS1 locus. Four type 2 diabetes-associated islet hyper-DMRs 31 overlapping with TKO hyper-DMRs are highlighted in green. A specific TKO hyper-DMR showing decreased ATAC, H3K4me1, and H3K27ac signals is highlighted in pink.
    Figure Legend Snippet: Pancreas-specific hyper-DMRs show reduced chromatin activity during pancreatic differentiation. a Volcano plot of WGBS data illustrating differentially methylated CpGs (DMCs) identified in TKO_PP cells compared with WT_PP cells. Red and blue represent increased and decreased 5mC in TKO_PP cells, respectively (credible methylation difference > 0.2). b Heatmap illustrating methylation difference between TKO_PP and WT_PP cells at centers of annotated genomic features (± 5 kb) for chromatin accessibility (ATAC), hydroxylation (5hmC), TF binding (FOXA2, GATA4, GATA6, and PDX1), bivalent promoters, poised enhancers, and active enhancers. Average 5mC signals of every 100-bp bin were calculated. c Classification of TKO hyper-DMRs based on 5mC levels in hESCs (green), WT_PP cells (blue), and TKO_PP cells (red). d Average plots of FOXA2 (left column), GATA4 (middle column), and GATA6 (right column) signal at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in pancreatic progenitors. e Average plots of ATAC (left column), H3K27ac (middle column), and H3K4me1 (right column) at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in WT_PP (blue) and TKO_PP (red) cells. f Average plots of 5mC at proximal (≤ 1 kb from TSS) and distal ( > 1 kb from TSS) decreased accessible regions in WT_PP (blue) and TKO_PP (red) cells. g Genome-browser view of the PDX1/PDX1-AS1 locus. Four type 2 diabetes-associated islet hyper-DMRs 31 overlapping with TKO hyper-DMRs are highlighted in green. A specific TKO hyper-DMR showing decreased ATAC, H3K4me1, and H3K27ac signals is highlighted in pink.

    Techniques Used: Activity Assay, Methylation, Binding Assay

    5) Product Images from "Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation"

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

    Journal: Cancer Medicine

    doi: 10.1002/cam4.830

    DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.
    Figure Legend Snippet: DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Techniques Used: Expressing, Genomic Sequencing, Methylation

    6) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )
    Figure Legend Snippet: DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )

    Techniques Used: Mouse Assay, DNA Methylation Assay, Polymerase Chain Reaction, BAC Assay, Isolation, CTL Assay, Methylation

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    7) Product Images from "Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients"

    Article Title: Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2019/8156592

    Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p
    Figure Legend Snippet: Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p

    Techniques Used: Sequencing, Modification, Real-time Polymerase Chain Reaction, Amplification

    8) Product Images from "Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome"

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome

    Journal: Genome Biology

    doi: 10.1186/gb-2012-13-10-r93

    5hmC profiling of mouse liver DNA . (a) An 11 kb promoter array region split into six indicated regions for epigenetic mapping analysis. (b) 5hmC and 5mC enrichment peaks in liver DNA map largely to intra-genic regions: left, distribution of all array probes; right, 5hmC and 5mC enrichment peaks. Chi 2 values indicate significance of the peak distributions compared to distribution of all array probes. (c) EpiMark qPCR of hmCpG (purple), 5mCpG (red) and non-modified CpG (green) levels over loci in control livers (n = 2). Percentage scores represent frequency of each CpG state over a single Msp I site. '5hmC +ve', 5hmC-positive regions; '5hmC -ve', 5hmC-negative regions. Error bars represent standard errors. (d) Box plot showing levels of 5hmC (purple) and 5mC (red) over 1 kb long enhancer and promoter regions. Asterisk denotes significant difference in signal levels ( P
    Figure Legend Snippet: 5hmC profiling of mouse liver DNA . (a) An 11 kb promoter array region split into six indicated regions for epigenetic mapping analysis. (b) 5hmC and 5mC enrichment peaks in liver DNA map largely to intra-genic regions: left, distribution of all array probes; right, 5hmC and 5mC enrichment peaks. Chi 2 values indicate significance of the peak distributions compared to distribution of all array probes. (c) EpiMark qPCR of hmCpG (purple), 5mCpG (red) and non-modified CpG (green) levels over loci in control livers (n = 2). Percentage scores represent frequency of each CpG state over a single Msp I site. '5hmC +ve', 5hmC-positive regions; '5hmC -ve', 5hmC-negative regions. Error bars represent standard errors. (d) Box plot showing levels of 5hmC (purple) and 5mC (red) over 1 kb long enhancer and promoter regions. Asterisk denotes significant difference in signal levels ( P

    Techniques Used: Real-time Polymerase Chain Reaction, Modification

    9) Product Images from "Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression"

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw316

    Validation of discordant CCGG sites by EpiMark 5-hmC and 5-mC Analysis kit. ( A ) CCGG sites with hmC values discordant for more than 0.2 between TAB-Methyl-SEQ and TrueMethyl-WGBS data were validated by qPCR ( n = 24). All sites were covered by at least 25 reads in both NGS experiments. ( B ) CCGG sites with hmC values discordant for more than 0.2 between TAB-450K and TrueMethyl-450K data were validated by qPCR ( n = 23). For the coordinates of these 47 CpG sites and the corresponding primers sequences see Supplementary Table S1. The axes scales are from 0.0 (no hmC at given CCGG site) to 1.0 (all cytosine residues are represented by hmC).
    Figure Legend Snippet: Validation of discordant CCGG sites by EpiMark 5-hmC and 5-mC Analysis kit. ( A ) CCGG sites with hmC values discordant for more than 0.2 between TAB-Methyl-SEQ and TrueMethyl-WGBS data were validated by qPCR ( n = 24). All sites were covered by at least 25 reads in both NGS experiments. ( B ) CCGG sites with hmC values discordant for more than 0.2 between TAB-450K and TrueMethyl-450K data were validated by qPCR ( n = 23). For the coordinates of these 47 CpG sites and the corresponding primers sequences see Supplementary Table S1. The axes scales are from 0.0 (no hmC at given CCGG site) to 1.0 (all cytosine residues are represented by hmC).

    Techniques Used: Real-time Polymerase Chain Reaction, Next-Generation Sequencing

    10) Product Images from "Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation"

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    doi: 10.1002/jbmr.2711

    Chondroprogenitor differentiation in vitro is accompanied by an increase in global 5hmC levels . A. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to Sox9 (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. B. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to 5hmC (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. Insets show higher magnification of selected areas. C. Representative immunoblot of 5hmC and 5mC levels during chondrogenic differentiation of ATDC5 cells. DNA isolated from cells undergoing differentiation at the indicated time points, was probed with antibodies specific to 5hmC, 5mC and single stranded DNA (ssDNA, as a control for loading). D. Quantification of the modified cytosine levels as represented in the immunoblot in B, normalized to ssDNA. Data represented as mean ± SD from three independent biological replicates (n = 3). * denotes p
    Figure Legend Snippet: Chondroprogenitor differentiation in vitro is accompanied by an increase in global 5hmC levels . A. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to Sox9 (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. B. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to 5hmC (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. Insets show higher magnification of selected areas. C. Representative immunoblot of 5hmC and 5mC levels during chondrogenic differentiation of ATDC5 cells. DNA isolated from cells undergoing differentiation at the indicated time points, was probed with antibodies specific to 5hmC, 5mC and single stranded DNA (ssDNA, as a control for loading). D. Quantification of the modified cytosine levels as represented in the immunoblot in B, normalized to ssDNA. Data represented as mean ± SD from three independent biological replicates (n = 3). * denotes p

    Techniques Used: In Vitro, Immunostaining, Isolation, Modification

    Distinct changes in global 5-hydroxymethylcytosine (5hmC) are associated with different stages of chondrogenic differentiation during embryonic limb development . A. Scheme demonstrating the different pathways and enzyme families responsible for 5hmC generation and turnover. DNA methyltransferases (DNMT) methylate cytosine residues at the C-5 carbon to produce 5-methylcytosine (5mC). DNMT1 is responsible for the maintenance of cytosine methylation marks during cell division, whereas DNMT3A and 3B establish de novo cytosine methylation. The TET family of enzymes (TETs) including TET 1, 2 and 3 convert 5mC to 5-hydroxymethylcytosines (5hmC) and further oxidized products 5-formylcytosines (5fC) and 5-carboxylcyosines (5caC) that can be acted upon by the Base-Excision Repair (BER) glycosylase, TDG. B. Immunostaining of the developing mouse tibial anlagen at embryonic days E11.5 (days post coitus), and growth plate at E13.5 and E17.5 with antibodies specific to 5hmC and Sox9 (red). Nuclei (blue) are counterstained with DAPI. Scale bar = 50µm.
    Figure Legend Snippet: Distinct changes in global 5-hydroxymethylcytosine (5hmC) are associated with different stages of chondrogenic differentiation during embryonic limb development . A. Scheme demonstrating the different pathways and enzyme families responsible for 5hmC generation and turnover. DNA methyltransferases (DNMT) methylate cytosine residues at the C-5 carbon to produce 5-methylcytosine (5mC). DNMT1 is responsible for the maintenance of cytosine methylation marks during cell division, whereas DNMT3A and 3B establish de novo cytosine methylation. The TET family of enzymes (TETs) including TET 1, 2 and 3 convert 5mC to 5-hydroxymethylcytosines (5hmC) and further oxidized products 5-formylcytosines (5fC) and 5-carboxylcyosines (5caC) that can be acted upon by the Base-Excision Repair (BER) glycosylase, TDG. B. Immunostaining of the developing mouse tibial anlagen at embryonic days E11.5 (days post coitus), and growth plate at E13.5 and E17.5 with antibodies specific to 5hmC and Sox9 (red). Nuclei (blue) are counterstained with DAPI. Scale bar = 50µm.

    Techniques Used: Methylation, Immunostaining

    11) Product Images from "Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation"

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    doi: 10.1002/jbmr.2711

    Chondroprogenitor differentiation in vitro is accompanied by an increase in global 5hmC levels . A. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to Sox9 (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. B. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to 5hmC (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. Insets show higher magnification of selected areas. C. Representative immunoblot of 5hmC and 5mC levels during chondrogenic differentiation of ATDC5 cells. DNA isolated from cells undergoing differentiation at the indicated time points, was probed with antibodies specific to 5hmC, 5mC and single stranded DNA (ssDNA, as a control for loading). D. Quantification of the modified cytosine levels as represented in the immunoblot in B, normalized to ssDNA. Data represented as mean ± SD from three independent biological replicates (n = 3). * denotes p
    Figure Legend Snippet: Chondroprogenitor differentiation in vitro is accompanied by an increase in global 5hmC levels . A. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to Sox9 (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. B. Immunostaining of ATDC5 cells over the course of chondrogenesis from progenitor cells (D0 = day 0) to mature chondrocyte (D20 = day 20) with an antibody specific to 5hmC (red). Nuclei (blue) are counterstained with DAPI; merge (violet) is shown in the bottom panel. Scale bar = 30µm. Insets show higher magnification of selected areas. C. Representative immunoblot of 5hmC and 5mC levels during chondrogenic differentiation of ATDC5 cells. DNA isolated from cells undergoing differentiation at the indicated time points, was probed with antibodies specific to 5hmC, 5mC and single stranded DNA (ssDNA, as a control for loading). D. Quantification of the modified cytosine levels as represented in the immunoblot in B, normalized to ssDNA. Data represented as mean ± SD from three independent biological replicates (n = 3). * denotes p

    Techniques Used: In Vitro, Immunostaining, Isolation, Modification

    Distinct changes in global 5-hydroxymethylcytosine (5hmC) are associated with different stages of chondrogenic differentiation during embryonic limb development . A. Scheme demonstrating the different pathways and enzyme families responsible for 5hmC generation and turnover. DNA methyltransferases (DNMT) methylate cytosine residues at the C-5 carbon to produce 5-methylcytosine (5mC). DNMT1 is responsible for the maintenance of cytosine methylation marks during cell division, whereas DNMT3A and 3B establish de novo cytosine methylation. The TET family of enzymes (TETs) including TET 1, 2 and 3 convert 5mC to 5-hydroxymethylcytosines (5hmC) and further oxidized products 5-formylcytosines (5fC) and 5-carboxylcyosines (5caC) that can be acted upon by the Base-Excision Repair (BER) glycosylase, TDG. B. Immunostaining of the developing mouse tibial anlagen at embryonic days E11.5 (days post coitus), and growth plate at E13.5 and E17.5 with antibodies specific to 5hmC and Sox9 (red). Nuclei (blue) are counterstained with DAPI. Scale bar = 50µm.
    Figure Legend Snippet: Distinct changes in global 5-hydroxymethylcytosine (5hmC) are associated with different stages of chondrogenic differentiation during embryonic limb development . A. Scheme demonstrating the different pathways and enzyme families responsible for 5hmC generation and turnover. DNA methyltransferases (DNMT) methylate cytosine residues at the C-5 carbon to produce 5-methylcytosine (5mC). DNMT1 is responsible for the maintenance of cytosine methylation marks during cell division, whereas DNMT3A and 3B establish de novo cytosine methylation. The TET family of enzymes (TETs) including TET 1, 2 and 3 convert 5mC to 5-hydroxymethylcytosines (5hmC) and further oxidized products 5-formylcytosines (5fC) and 5-carboxylcyosines (5caC) that can be acted upon by the Base-Excision Repair (BER) glycosylase, TDG. B. Immunostaining of the developing mouse tibial anlagen at embryonic days E11.5 (days post coitus), and growth plate at E13.5 and E17.5 with antibodies specific to 5hmC and Sox9 (red). Nuclei (blue) are counterstained with DAPI. Scale bar = 50µm.

    Techniques Used: Methylation, Immunostaining

    12) Product Images from "Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients"

    Article Title: Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2019/8156592

    Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p
    Figure Legend Snippet: Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p

    Techniques Used: Sequencing, Modification, Real-time Polymerase Chain Reaction, Amplification

    13) Product Images from "Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia"

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0252-7

    Genome-wide distribution of 5-hmC and 5-mC over CGIs, shores, shelfs, enhancers and promoters. a Global levels of 5-hmC, 5-mC, H3K4me1 and H3K27ac coverage over CGIs, shores and shelfs in CLL samples. b Global levels of 5-hmC and 5-mC coverage over CGIs, shores and shelfs in CLL samples. c , d Global levels of 5-hmC over H3K27ac peaks at active enhancers ( c ) and active promoters ( d ) in B-cells and CLL patient samples. Coverage is presented as normalized number of unique reads per bp per peak
    Figure Legend Snippet: Genome-wide distribution of 5-hmC and 5-mC over CGIs, shores, shelfs, enhancers and promoters. a Global levels of 5-hmC, 5-mC, H3K4me1 and H3K27ac coverage over CGIs, shores and shelfs in CLL samples. b Global levels of 5-hmC and 5-mC coverage over CGIs, shores and shelfs in CLL samples. c , d Global levels of 5-hmC over H3K27ac peaks at active enhancers ( c ) and active promoters ( d ) in B-cells and CLL patient samples. Coverage is presented as normalized number of unique reads per bp per peak

    Techniques Used: Genome Wide

    Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p
    Figure Legend Snippet: Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p

    Techniques Used: Functional Assay, Expressing, Negative Control, Transfection, MTT Assay

    Mass spectrometry data for global 5-hmC and 5-mC levels and distribution of peaks across the genome. a Absolute levels of 5-hmC and 5-mC in CLL B-cells and normal B-cells, b absolute levels of 5-hmC and 5-mC in CLL subtypes and B-cell subsets and c DMR and DhMR peak distribution of uniquely mapped reads for M-CLL versus normal memory B-cell and UCLL versus normal naive B-cell comparisons across the genome
    Figure Legend Snippet: Mass spectrometry data for global 5-hmC and 5-mC levels and distribution of peaks across the genome. a Absolute levels of 5-hmC and 5-mC in CLL B-cells and normal B-cells, b absolute levels of 5-hmC and 5-mC in CLL subtypes and B-cell subsets and c DMR and DhMR peak distribution of uniquely mapped reads for M-CLL versus normal memory B-cell and UCLL versus normal naive B-cell comparisons across the genome

    Techniques Used: Mass Spectrometry

    Genome-wide patterns of 5-hmC and 5-mC distribution over highly, poorly and not expressed genes. a – c shows the global 5-hmC and 5-mC distribution over gene-body of highly ( a ), poorly ( b ) and not ( c ) expressed genes. Left side plots numbered as (i) shows the data for CLL samples and the right side plots numbered with (ii) shows the data for normal B cells
    Figure Legend Snippet: Genome-wide patterns of 5-hmC and 5-mC distribution over highly, poorly and not expressed genes. a – c shows the global 5-hmC and 5-mC distribution over gene-body of highly ( a ), poorly ( b ) and not ( c ) expressed genes. Left side plots numbered as (i) shows the data for CLL samples and the right side plots numbered with (ii) shows the data for normal B cells

    Techniques Used: Genome Wide

    Genome-wide distribution of 5-hmC and 5-mC over the promoters and gene-body. a Global 5-hmC and 5-mC distribution pattern over gene-body in B-cell subsets, b global 5-hmC and 5-mC distribution pattern over gene-body in total CLL patient samples and normal B-cells
    Figure Legend Snippet: Genome-wide distribution of 5-hmC and 5-mC over the promoters and gene-body. a Global 5-hmC and 5-mC distribution pattern over gene-body in B-cell subsets, b global 5-hmC and 5-mC distribution pattern over gene-body in total CLL patient samples and normal B-cells

    Techniques Used: Genome Wide

    14) Product Images from "Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine"

    Article Title: Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku216

    Boronic acid (BA) specifically inhibit the amplification activity of Taq DNA polymerase on 5ghmC-containing dsDNA. ( a ) The sequences of oligonucleotides containing modified cytosines, where X indicates C, 5mC 5hmC or 5ghmC, and the underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) The qPCR curves of 5ghmC-ds100mer in the presence (dashed line) or absence (solid line) of BA. ( c ) The qPCR curves of ds100mers containing a C (black solid line), 5mC (black dashed line), 5hmC (black dense dashed line) or 5ghmC (gray dashed line) at the 58th nucleotide in the absence of any BAs; gray solid lines indicate the PCR products of NTC (No Template Control). The solid straight lines indicate the defined threshold of qPCR. ( d ) The qPCR curves of C-ds100mer, 5mC-ds100mer and unglucosylated 5hmC-ds100mer in the presence of BA (followed by the gray solid line, gray dashed line and gray dense dashed line) or absence of BA (followed by the black solid line, black dashed line and black dense dashed line).
    Figure Legend Snippet: Boronic acid (BA) specifically inhibit the amplification activity of Taq DNA polymerase on 5ghmC-containing dsDNA. ( a ) The sequences of oligonucleotides containing modified cytosines, where X indicates C, 5mC 5hmC or 5ghmC, and the underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) The qPCR curves of 5ghmC-ds100mer in the presence (dashed line) or absence (solid line) of BA. ( c ) The qPCR curves of ds100mers containing a C (black solid line), 5mC (black dashed line), 5hmC (black dense dashed line) or 5ghmC (gray dashed line) at the 58th nucleotide in the absence of any BAs; gray solid lines indicate the PCR products of NTC (No Template Control). The solid straight lines indicate the defined threshold of qPCR. ( d ) The qPCR curves of C-ds100mer, 5mC-ds100mer and unglucosylated 5hmC-ds100mer in the presence of BA (followed by the gray solid line, gray dashed line and gray dense dashed line) or absence of BA (followed by the black solid line, black dashed line and black dense dashed line).

    Techniques Used: Amplification, Activity Assay, Modification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    The effects of four BA derivatives on Taq DNA polymerase replicating 5ghmC-ds100mer. ( a ) qPCR curves in the presence (dashed line) or absence (solid line) of BA derivatives. ( b ) Δ C t values for C-, 5mC-, 5hmC- and 5ghmC-containing DNA in the presence and absence of BA derivations. Δ C t is the difference between the C t value with BA derivatives and without BA derivatives. Four BA derivatives are shown 2-CB-PBA (2-(2'-chlorobenzyloxy) phenylboronic acid), 3-CPBA (3-chlorophenylboronic acid), PBA (phenyl-boronic acid) and 3-D-PBA (3-(Dansylamino) phenylboronic acid) from top to bottom. Error bars represent the standard deviation from the mean of three independent experiments.
    Figure Legend Snippet: The effects of four BA derivatives on Taq DNA polymerase replicating 5ghmC-ds100mer. ( a ) qPCR curves in the presence (dashed line) or absence (solid line) of BA derivatives. ( b ) Δ C t values for C-, 5mC-, 5hmC- and 5ghmC-containing DNA in the presence and absence of BA derivations. Δ C t is the difference between the C t value with BA derivatives and without BA derivatives. Four BA derivatives are shown 2-CB-PBA (2-(2'-chlorobenzyloxy) phenylboronic acid), 3-CPBA (3-chlorophenylboronic acid), PBA (phenyl-boronic acid) and 3-D-PBA (3-(Dansylamino) phenylboronic acid) from top to bottom. Error bars represent the standard deviation from the mean of three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

    15) Product Images from "Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism"

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1090

    The conversion of 5mC to 5hmC by redox-active quinones at cellular level. ( A and B ) UHPLC-MRM MS/MS analysis (A) and quantitation (B) of 5hmC in the enzymatic digest of genomic DNA from MRC-5 cells. The cells were treated with 20 μM TCBQ or TCHQ. The peak corresponding to 5hmC is denoted by the arrow, and the other two peaks are un-identified. ( C ) Immunodotblotting analysis of 5hmC in MRC-5 cells upon 20 μM TCBQ- or TCHQ-treatment (left panel). The amount of uploaded DNA samples was indicated by methyl blue staining (right panel). N.C and P.C. represent negative and positive control, respectively. Control genomic DNA was extracted from the cells only treated with the solvent DMSO (0.02%, v/v) with the same amount as that used in TCBQ/TCHQ treatment. ** P
    Figure Legend Snippet: The conversion of 5mC to 5hmC by redox-active quinones at cellular level. ( A and B ) UHPLC-MRM MS/MS analysis (A) and quantitation (B) of 5hmC in the enzymatic digest of genomic DNA from MRC-5 cells. The cells were treated with 20 μM TCBQ or TCHQ. The peak corresponding to 5hmC is denoted by the arrow, and the other two peaks are un-identified. ( C ) Immunodotblotting analysis of 5hmC in MRC-5 cells upon 20 μM TCBQ- or TCHQ-treatment (left panel). The amount of uploaded DNA samples was indicated by methyl blue staining (right panel). N.C and P.C. represent negative and positive control, respectively. Control genomic DNA was extracted from the cells only treated with the solvent DMSO (0.02%, v/v) with the same amount as that used in TCBQ/TCHQ treatment. ** P

    Techniques Used: Mass Spectrometry, Quantitation Assay, Staining, Positive Control

    16) Product Images from "Cytosine modifications modulate the chromatin architecture of transcriptional enhancers"

    Article Title: Cytosine modifications modulate the chromatin architecture of transcriptional enhancers

    Journal: Genome Research

    doi: 10.1101/gr.211466.116

    De novo hydroxymethylated enhancers bind pioneer factors driving NPC commitment. ( A ) Average kinetic profiles of 5hmC, 5mC, H3K4me1, H3K27ac, and chromatin opening (FAIRE) at 20,492 regions gaining 5hmC (up-DhMRs) at 48 h of P19 ECC differentiation. ( B ) Average profiles of FOXA1, PBX1, and MEIS1 binding at up-DhMRs at 48 h of differentiation. ( C ) Heat map representations of 5hmC signal at MEIS1, PBX1, and FOXA1 enriched regions in 48 h RA-treated P19 cells.
    Figure Legend Snippet: De novo hydroxymethylated enhancers bind pioneer factors driving NPC commitment. ( A ) Average kinetic profiles of 5hmC, 5mC, H3K4me1, H3K27ac, and chromatin opening (FAIRE) at 20,492 regions gaining 5hmC (up-DhMRs) at 48 h of P19 ECC differentiation. ( B ) Average profiles of FOXA1, PBX1, and MEIS1 binding at up-DhMRs at 48 h of differentiation. ( C ) Heat map representations of 5hmC signal at MEIS1, PBX1, and FOXA1 enriched regions in 48 h RA-treated P19 cells.

    Techniques Used: Binding Assay

    17) Product Images from "Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression"

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw316

    Validation of discordant CCGG sites by EpiMark 5-hmC and 5-mC Analysis kit. ( A ) CCGG sites with hmC values discordant for more than 0.2 between TAB-Methyl-SEQ and TrueMethyl-WGBS data were validated by qPCR ( n = 24). All sites were covered by at least 25 reads in both NGS experiments. ( B ) CCGG sites with hmC values discordant for more than 0.2 between TAB-450K and TrueMethyl-450K data were validated by qPCR ( n = 23). For the coordinates of these 47 CpG sites and the corresponding primers sequences see Supplementary Table S1. The axes scales are from 0.0 (no hmC at given CCGG site) to 1.0 (all cytosine residues are represented by hmC).
    Figure Legend Snippet: Validation of discordant CCGG sites by EpiMark 5-hmC and 5-mC Analysis kit. ( A ) CCGG sites with hmC values discordant for more than 0.2 between TAB-Methyl-SEQ and TrueMethyl-WGBS data were validated by qPCR ( n = 24). All sites were covered by at least 25 reads in both NGS experiments. ( B ) CCGG sites with hmC values discordant for more than 0.2 between TAB-450K and TrueMethyl-450K data were validated by qPCR ( n = 23). For the coordinates of these 47 CpG sites and the corresponding primers sequences see Supplementary Table S1. The axes scales are from 0.0 (no hmC at given CCGG site) to 1.0 (all cytosine residues are represented by hmC).

    Techniques Used: Real-time Polymerase Chain Reaction, Next-Generation Sequencing

    18) Product Images from "Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum"

    Article Title: Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2014.87

    The mean increase in 5-hmC levels in the EN-2 promoter in autism compared control cerebellar samples using two independent assays: a shows the relative increase using the Epimark assay and b shows similar increase using the hMeDIP assay (means±s.d.). Panel c shows the highly significant positive association between 5-hmC and 5-mC ( P =0.002).
    Figure Legend Snippet: The mean increase in 5-hmC levels in the EN-2 promoter in autism compared control cerebellar samples using two independent assays: a shows the relative increase using the Epimark assay and b shows similar increase using the hMeDIP assay (means±s.d.). Panel c shows the highly significant positive association between 5-hmC and 5-mC ( P =0.002).

    Techniques Used:

    Global alterations in cerebellar levels of 5-mC ( a ), 5-hmC ( b ), DNA methyltransferases DNMT1, DNMT3A and DNMT3B ( c ), TET1, TET2, TET3 ( d ) and 8-oxo-dG ( e ) in 13 autism (ASD) and 13 matched control cerebellar samples. Results are expressed as the mean and s.d.'s with associated P -values.
    Figure Legend Snippet: Global alterations in cerebellar levels of 5-mC ( a ), 5-hmC ( b ), DNA methyltransferases DNMT1, DNMT3A and DNMT3B ( c ), TET1, TET2, TET3 ( d ) and 8-oxo-dG ( e ) in 13 autism (ASD) and 13 matched control cerebellar samples. Results are expressed as the mean and s.d.'s with associated P -values.

    Techniques Used:

    Panels a , b depict the correlations between 5-mC and 5-hmC in matched control and autism cerebellar samples, respectively, with associated correlation coefficients and P -values. Panels c , d show the correlation between 5-hmC and 8-oxo-dG in control and autism samples, respectively, with no correlation within the control samples and marginally significant positive association ( P =0.08) in the autism samples.
    Figure Legend Snippet: Panels a , b depict the correlations between 5-mC and 5-hmC in matched control and autism cerebellar samples, respectively, with associated correlation coefficients and P -values. Panels c , d show the correlation between 5-hmC and 8-oxo-dG in control and autism samples, respectively, with no correlation within the control samples and marginally significant positive association ( P =0.08) in the autism samples.

    Techniques Used:

    19) Product Images from "Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia"

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0252-7

    Genome-wide distribution of 5-hmC and 5-mC over CGIs, shores, shelfs, enhancers and promoters. a Global levels of 5-hmC, 5-mC, H3K4me1 and H3K27ac coverage over CGIs, shores and shelfs in CLL samples. b Global levels of 5-hmC and 5-mC coverage over CGIs, shores and shelfs in CLL samples. c , d Global levels of 5-hmC over H3K27ac peaks at active enhancers ( c ) and active promoters ( d ) in B-cells and CLL patient samples. Coverage is presented as normalized number of unique reads per bp per peak
    Figure Legend Snippet: Genome-wide distribution of 5-hmC and 5-mC over CGIs, shores, shelfs, enhancers and promoters. a Global levels of 5-hmC, 5-mC, H3K4me1 and H3K27ac coverage over CGIs, shores and shelfs in CLL samples. b Global levels of 5-hmC and 5-mC coverage over CGIs, shores and shelfs in CLL samples. c , d Global levels of 5-hmC over H3K27ac peaks at active enhancers ( c ) and active promoters ( d ) in B-cells and CLL patient samples. Coverage is presented as normalized number of unique reads per bp per peak

    Techniques Used: Genome Wide

    Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p
    Figure Legend Snippet: Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p

    Techniques Used: Functional Assay, Expressing, Negative Control, Transfection, MTT Assay

    Mass spectrometry data for global 5-hmC and 5-mC levels and distribution of peaks across the genome. a Absolute levels of 5-hmC and 5-mC in CLL B-cells and normal B-cells, b absolute levels of 5-hmC and 5-mC in CLL subtypes and B-cell subsets and c DMR and DhMR peak distribution of uniquely mapped reads for M-CLL versus normal memory B-cell and UCLL versus normal naive B-cell comparisons across the genome
    Figure Legend Snippet: Mass spectrometry data for global 5-hmC and 5-mC levels and distribution of peaks across the genome. a Absolute levels of 5-hmC and 5-mC in CLL B-cells and normal B-cells, b absolute levels of 5-hmC and 5-mC in CLL subtypes and B-cell subsets and c DMR and DhMR peak distribution of uniquely mapped reads for M-CLL versus normal memory B-cell and UCLL versus normal naive B-cell comparisons across the genome

    Techniques Used: Mass Spectrometry

    Genome-wide patterns of 5-hmC and 5-mC distribution over highly, poorly and not expressed genes. a – c shows the global 5-hmC and 5-mC distribution over gene-body of highly ( a ), poorly ( b ) and not ( c ) expressed genes. Left side plots numbered as (i) shows the data for CLL samples and the right side plots numbered with (ii) shows the data for normal B cells
    Figure Legend Snippet: Genome-wide patterns of 5-hmC and 5-mC distribution over highly, poorly and not expressed genes. a – c shows the global 5-hmC and 5-mC distribution over gene-body of highly ( a ), poorly ( b ) and not ( c ) expressed genes. Left side plots numbered as (i) shows the data for CLL samples and the right side plots numbered with (ii) shows the data for normal B cells

    Techniques Used: Genome Wide

    Genome-wide distribution of 5-hmC and 5-mC over the promoters and gene-body. a Global 5-hmC and 5-mC distribution pattern over gene-body in B-cell subsets, b global 5-hmC and 5-mC distribution pattern over gene-body in total CLL patient samples and normal B-cells
    Figure Legend Snippet: Genome-wide distribution of 5-hmC and 5-mC over the promoters and gene-body. a Global 5-hmC and 5-mC distribution pattern over gene-body in B-cell subsets, b global 5-hmC and 5-mC distribution pattern over gene-body in total CLL patient samples and normal B-cells

    Techniques Used: Genome Wide

    20) Product Images from "Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia"

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0252-7

    Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p
    Figure Legend Snippet: Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p

    Techniques Used: Functional Assay, Expressing, Negative Control, Transfection, MTT Assay

    21) Product Images from "Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia"

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0252-7

    Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p
    Figure Legend Snippet: Functional relevance of 5-hmC in regulating gene expression levels. a , b 5-hmC levels of selected 5hDMR genes in HG3 and MEC1 CLL cell lines respectively. TSH2B gene was used as the negative control for hMeDIP as provided by the kit. c Log10-fold change of 5-hmC and 5-mC levels of HG3 TET1/TET2siRNA samples over control siRNA samples d Log10-fold change of relative gene expression levels over GAPDH in HG3 TET1/TET2 siRNA samples over control siRNA samples. e Percentage of 5-hmC levels for sorted B-CLL samples compared to normal B cell samples using quantitative epimark 5-hmC and 5-mC analysis Kit. f Percentage of proliferation for NSMCE1, TUBGCP6 and TUBGCP3 siRNA transfected HG3 samples compared to control siRNA sample using MTT assay. *Indicates p

    Techniques Used: Functional Assay, Expressing, Negative Control, Transfection, MTT Assay

    22) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )
    Figure Legend Snippet: DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )

    Techniques Used: Mouse Assay, DNA Methylation Assay, Polymerase Chain Reaction, BAC Assay, Isolation, CTL Assay, Methylation

    DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p
    Figure Legend Snippet: DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p

    Techniques Used: DNA Methylation Assay, Derivative Assay, Stable Transfection, Expressing, shRNA, CTL Assay, Immunoprecipitation, Negative Control

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    C9ORF72 transcription decreases while a repressive histone methylation mark increases in the brain of C9-BAC mice during the first post-natal weeks. Values of human C9ORF72 in the BAC mouse cortex, normalized to the average of GAPDH and 18S, are shown for primers amplifying transcript variants V1, V2, V3 ( a ); V1, V3 ( b ) and V2 ( c ). Age groups are indicated on the x-axis in weeks (wks). Mean and standard error of the mean (SEM) are indicated by long and short bars respectively. For each primer set, a one-way analysis of variance was performed ( p
    Figure Legend Snippet: C9ORF72 transcription decreases while a repressive histone methylation mark increases in the brain of C9-BAC mice during the first post-natal weeks. Values of human C9ORF72 in the BAC mouse cortex, normalized to the average of GAPDH and 18S, are shown for primers amplifying transcript variants V1, V2, V3 ( a ); V1, V3 ( b ) and V2 ( c ). Age groups are indicated on the x-axis in weeks (wks). Mean and standard error of the mean (SEM) are indicated by long and short bars respectively. For each primer set, a one-way analysis of variance was performed ( p

    Techniques Used: Methylation, BAC Assay, Mouse Assay

    23) Product Images from "TET2 Mutations Are Associated with Specific 5-Methylcytosine and 5-Hydroxymethylcytosine Profiles in Patients with Chronic Myelomonocytic Leukemia"

    Article Title: TET2 Mutations Are Associated with Specific 5-Methylcytosine and 5-Hydroxymethylcytosine Profiles in Patients with Chronic Myelomonocytic Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031605

    Analysis of 5 hmC and 5 mC levels in genes hypermethylated in CMML TET2-mut in comparison to TET2-wt patients. The percentage of 5 hmC, 5 mC and ratio between 5 hmC/5 mC were measured in 8 CMML  TET2 -mut and 5  TET2 -wt patient samples using qPCR. Two CpG located 5′upstream (−38 and −244 bp respectively) to the CpG analyzed in the methylation array in the case of  LAX1 ; one upstream CpG (59 bp) and another downstream CpG (58 bp) to the CpG in the case of  SLC22A12  gene and one CpG downstream (5 bp) in the case of  VHL  gene were analyzed. Median values of percentage of 5 hmC, 5 mC or ratio between 5 hmC/5 mC are indicated and P values were obtained using the 2-tailed T test or U Mann Whitney test. CG: CpG dinucleotide included in the array; CCGG: CpG dinucleotide in which 5 hmC and 5 mC have been analysed and TSS: transcriptional start site.
    Figure Legend Snippet: Analysis of 5 hmC and 5 mC levels in genes hypermethylated in CMML TET2-mut in comparison to TET2-wt patients. The percentage of 5 hmC, 5 mC and ratio between 5 hmC/5 mC were measured in 8 CMML TET2 -mut and 5 TET2 -wt patient samples using qPCR. Two CpG located 5′upstream (−38 and −244 bp respectively) to the CpG analyzed in the methylation array in the case of LAX1 ; one upstream CpG (59 bp) and another downstream CpG (58 bp) to the CpG in the case of SLC22A12 gene and one CpG downstream (5 bp) in the case of VHL gene were analyzed. Median values of percentage of 5 hmC, 5 mC or ratio between 5 hmC/5 mC are indicated and P values were obtained using the 2-tailed T test or U Mann Whitney test. CG: CpG dinucleotide included in the array; CCGG: CpG dinucleotide in which 5 hmC and 5 mC have been analysed and TSS: transcriptional start site.

    Techniques Used: Real-time Polymerase Chain Reaction, Methylation, MANN-WHITNEY

    24) Product Images from "Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation"

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

    Journal: Cancer Medicine

    doi: 10.1002/cam4.830

    DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.
    Figure Legend Snippet: DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Techniques Used: Expressing, Genomic Sequencing, Methylation

    25) Product Images from "TET1 inhibits gastric cancer growth and metastasis by PTEN demethylation and re-expression"

    Article Title: TET1 inhibits gastric cancer growth and metastasis by PTEN demethylation and re-expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8900

    TET1 is down-regulated in gastric cancer A. TET1, B. TET2 and C. TET3 mRNA expression in gastric cancer tissues verse adjacent non-tumor tissues. Data are showed as log 2 fold changes (tumor/normal). Results were analyzed by the Wilcoxon Signed Ranks test. D. 5-hmC and 5-mC content were detected by dot blot in gastric cancer tissues compared to adjacent normal tissues. E. IHC analysis of TET1 in gastric cancer tissues and adjacent non-tumor tissues (upper panel 20×, lower panel 200×). F. Kaplan–Meier analysis of overall survival in 80 patients with gastric cancer.
    Figure Legend Snippet: TET1 is down-regulated in gastric cancer A. TET1, B. TET2 and C. TET3 mRNA expression in gastric cancer tissues verse adjacent non-tumor tissues. Data are showed as log 2 fold changes (tumor/normal). Results were analyzed by the Wilcoxon Signed Ranks test. D. 5-hmC and 5-mC content were detected by dot blot in gastric cancer tissues compared to adjacent normal tissues. E. IHC analysis of TET1 in gastric cancer tissues and adjacent non-tumor tissues (upper panel 20×, lower panel 200×). F. Kaplan–Meier analysis of overall survival in 80 patients with gastric cancer.

    Techniques Used: Expressing, Dot Blot, Immunohistochemistry

    26) Product Images from "Rapamycin ameliorates chitosan nanoparticle-induced developmental defects of preimplantation embryos in mice"

    Article Title: Rapamycin ameliorates chitosan nanoparticle-induced developmental defects of preimplantation embryos in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10813

    CSNPs-altered epigenetics in preimplantation embryos A . Real time qRT-PCR analysis of the Tet family (Tet-1, Tet-2, Tet-3) and B . the Dnmt family (Dnmt1, Dnmt3a, Dnmt3b). C . 5-mC to 5-hmC analysis. D . Immunostaining of 5-mC and 5-hmC. The fluorescence intensities of 5-mC and 5-hmC were measured in both the ICM and the TE. E . Heterochromatin condensation was observed in the control and CNSPs-treated groups using TEM. To calculate each type of heterochromatin and based on the condensation, we divided the embryos into 3 groups: Types I, II and III. Transcriptional silencing is associated with the targeting of genomic sequences to repressive (heterochromatic) nuclear compartments. F . The H3K27me3 expression level was analyzed using immunofluorescence staining. Embryos were divided into Types I, II, and III by their expression patterns. G . The relative expression levels of methylase (Ezh2) and its co-factors (Eed5, Suz12) were measured using real time qRT-PCR analysis. *, **, and NS indicate p
    Figure Legend Snippet: CSNPs-altered epigenetics in preimplantation embryos A . Real time qRT-PCR analysis of the Tet family (Tet-1, Tet-2, Tet-3) and B . the Dnmt family (Dnmt1, Dnmt3a, Dnmt3b). C . 5-mC to 5-hmC analysis. D . Immunostaining of 5-mC and 5-hmC. The fluorescence intensities of 5-mC and 5-hmC were measured in both the ICM and the TE. E . Heterochromatin condensation was observed in the control and CNSPs-treated groups using TEM. To calculate each type of heterochromatin and based on the condensation, we divided the embryos into 3 groups: Types I, II and III. Transcriptional silencing is associated with the targeting of genomic sequences to repressive (heterochromatic) nuclear compartments. F . The H3K27me3 expression level was analyzed using immunofluorescence staining. Embryos were divided into Types I, II, and III by their expression patterns. G . The relative expression levels of methylase (Ezh2) and its co-factors (Eed5, Suz12) were measured using real time qRT-PCR analysis. *, **, and NS indicate p

    Techniques Used: Quantitative RT-PCR, Immunostaining, Fluorescence, Transmission Electron Microscopy, Genomic Sequencing, Expressing, Immunofluorescence, Staining

    27) Product Images from "TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes"

    Article Title: TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes

    Journal: Scientific Reports

    doi: 10.1038/srep42990

    DNA methylation and 5hmC status in the MMP3 promoter. ( A ) Immunohistochemistry analysis of human cartilage tissues. Cartilage sections were obtained from normal and OA-affected cartilages. Sections were stained with 5hmC anti body. Left scale bars, 500 μm. Right scale bar, 200 μm. ( B ) Bisulfite genomic sequencing results of the MMP3 promoter regions in TC28 cells after Ad-GFP or Ad-TWIST1 infection at 72 hours. Black, methylated; white, unmethylated. ( C ) To detect locus-specific 5hmC and 5mC status at CCGG site in MMP3 promoter, restriction enzyme digestion and quantitative polymerase chain reaction were performed. *P
    Figure Legend Snippet: DNA methylation and 5hmC status in the MMP3 promoter. ( A ) Immunohistochemistry analysis of human cartilage tissues. Cartilage sections were obtained from normal and OA-affected cartilages. Sections were stained with 5hmC anti body. Left scale bars, 500 μm. Right scale bar, 200 μm. ( B ) Bisulfite genomic sequencing results of the MMP3 promoter regions in TC28 cells after Ad-GFP or Ad-TWIST1 infection at 72 hours. Black, methylated; white, unmethylated. ( C ) To detect locus-specific 5hmC and 5mC status at CCGG site in MMP3 promoter, restriction enzyme digestion and quantitative polymerase chain reaction were performed. *P

    Techniques Used: DNA Methylation Assay, Immunohistochemistry, Staining, Genomic Sequencing, Infection, Methylation, Real-time Polymerase Chain Reaction

    28) Product Images from "α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis"

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0802-8

    CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p
    Figure Legend Snippet: CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Techniques Used: CRISPR, Activity Assay, Western Blot, Plasmid Preparation, Microscopy

    KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p
    Figure Legend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Techniques Used: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
    Article Snippet: .. To quantify the absolute levels of both 5hmC as well as 5mC over these regions, we used the EpiMark™ 5hmC and 5-mC Analysis Kit (New England BioLabs) followed by qPCR (Figure ; Additional file ; see Materials and methods). ..

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression
    Article Snippet: .. Validation of hmC values by qPCR Validation of BS and hmC calls at selected CCGG sites was done by EpiMark 5-hmC and 5-mC Analysis Kit (New England Biolabs, MA, USA). .. Three aliquots per sample were processed from 1–1.5 μg of input DNA: (i) fully untreated (positive control); (ii) βGT/MspI-treated (hmC signal) and (iii) MspI-treated (negative control).

    Quantitation Assay:

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: .. Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. ..

    DNA Sequencing:

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation
    Article Snippet: .. Validation of the enriched DNA sequencing results was performed using the EpiMark 5hmC and 5mC Analysis Kit (NEB) as per suppliers’ protocol. ..

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    New England Biolabs 5 mc analysis kit
    Figure 2. Genomic distribution of 5-hmC and <t>5-mC</t> sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation
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    Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation

    Journal: Epigenetics

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    doi: 10.4161/epi.24280

    Figure Lengend Snippet: Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation

    Article Snippet: The efficiency of glucosylation for hydoxymethylcytosines and enzyme digestion was confirmed using EpiMark 5-hmC and 5-mC Analysis Kit (NEB).

    Techniques: Methylation

    CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions.

    Techniques: CRISPR, Activity Assay, Western Blot, Plasmid Preparation, Microscopy

    KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions.

    Techniques: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

    TET1 is required for the PAX4 enhancer to achieve a hypomethylated state. a Genome-browser view of the PAX4 locus with increased methylation and decreased chromatin associability upon TET depletion at a TET1/FOXA2 co-bound region featuring enhancer signatures H3K4me1 and H3K27ac. b Locus-specific increase in 5mC at the PAX4 enhancer in TKO or TET1KO samples compared with TET2/3DKO samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation.

    Journal: bioRxiv

    Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

    doi: 10.1101/2020.05.20.107532

    Figure Lengend Snippet: TET1 is required for the PAX4 enhancer to achieve a hypomethylated state. a Genome-browser view of the PAX4 locus with increased methylation and decreased chromatin associability upon TET depletion at a TET1/FOXA2 co-bound region featuring enhancer signatures H3K4me1 and H3K27ac. b Locus-specific increase in 5mC at the PAX4 enhancer in TKO or TET1KO samples compared with TET2/3DKO samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation.

    Article Snippet: Locus-specific detection of 5mC Detection of 5mC content at particular CCGG sites was performed using the Epimark 5hmC and 5mC analysis kit (New England Biolabs) following the manufacturer’s instructions.

    Techniques: Methylation

    Pancreas-specific hyper-DMRs show reduced chromatin activity during pancreatic differentiation. a Volcano plot of WGBS data illustrating differentially methylated CpGs (DMCs) identified in TKO_PP cells compared with WT_PP cells. Red and blue represent increased and decreased 5mC in TKO_PP cells, respectively (credible methylation difference > 0.2). b Heatmap illustrating methylation difference between TKO_PP and WT_PP cells at centers of annotated genomic features (± 5 kb) for chromatin accessibility (ATAC), hydroxylation (5hmC), TF binding (FOXA2, GATA4, GATA6, and PDX1), bivalent promoters, poised enhancers, and active enhancers. Average 5mC signals of every 100-bp bin were calculated. c Classification of TKO hyper-DMRs based on 5mC levels in hESCs (green), WT_PP cells (blue), and TKO_PP cells (red). d Average plots of FOXA2 (left column), GATA4 (middle column), and GATA6 (right column) signal at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in pancreatic progenitors. e Average plots of ATAC (left column), H3K27ac (middle column), and H3K4me1 (right column) at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in WT_PP (blue) and TKO_PP (red) cells. f Average plots of 5mC at proximal (≤ 1 kb from TSS) and distal ( > 1 kb from TSS) decreased accessible regions in WT_PP (blue) and TKO_PP (red) cells. g Genome-browser view of the PDX1/PDX1-AS1 locus. Four type 2 diabetes-associated islet hyper-DMRs 31 overlapping with TKO hyper-DMRs are highlighted in green. A specific TKO hyper-DMR showing decreased ATAC, H3K4me1, and H3K27ac signals is highlighted in pink.

    Journal: bioRxiv

    Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

    doi: 10.1101/2020.05.20.107532

    Figure Lengend Snippet: Pancreas-specific hyper-DMRs show reduced chromatin activity during pancreatic differentiation. a Volcano plot of WGBS data illustrating differentially methylated CpGs (DMCs) identified in TKO_PP cells compared with WT_PP cells. Red and blue represent increased and decreased 5mC in TKO_PP cells, respectively (credible methylation difference > 0.2). b Heatmap illustrating methylation difference between TKO_PP and WT_PP cells at centers of annotated genomic features (± 5 kb) for chromatin accessibility (ATAC), hydroxylation (5hmC), TF binding (FOXA2, GATA4, GATA6, and PDX1), bivalent promoters, poised enhancers, and active enhancers. Average 5mC signals of every 100-bp bin were calculated. c Classification of TKO hyper-DMRs based on 5mC levels in hESCs (green), WT_PP cells (blue), and TKO_PP cells (red). d Average plots of FOXA2 (left column), GATA4 (middle column), and GATA6 (right column) signal at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in pancreatic progenitors. e Average plots of ATAC (left column), H3K27ac (middle column), and H3K4me1 (right column) at pancreas-specific hyper-DMRs or non-pancreatic hyper-DMRs in WT_PP (blue) and TKO_PP (red) cells. f Average plots of 5mC at proximal (≤ 1 kb from TSS) and distal ( > 1 kb from TSS) decreased accessible regions in WT_PP (blue) and TKO_PP (red) cells. g Genome-browser view of the PDX1/PDX1-AS1 locus. Four type 2 diabetes-associated islet hyper-DMRs 31 overlapping with TKO hyper-DMRs are highlighted in green. A specific TKO hyper-DMR showing decreased ATAC, H3K4me1, and H3K27ac signals is highlighted in pink.

    Article Snippet: Locus-specific detection of 5mC Detection of 5mC content at particular CCGG sites was performed using the Epimark 5hmC and 5mC analysis kit (New England Biolabs) following the manufacturer’s instructions.

    Techniques: Activity Assay, Methylation, Binding Assay

    DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Journal: Cancer Medicine

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

    doi: 10.1002/cam4.830

    Figure Lengend Snippet: DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region.

    Techniques: Expressing, Genomic Sequencing, Methylation