quest 5hmc detection kit  (Zymo Research)


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    Name:
    Quest 5 hmC Detection Kit
    Description:
    The Quest 5 hmC Detection Kit allows for locus specific detection of 5 hydroxymethylcytosine 5 hmC using a simple and efficient reaction setup Traditional methods cannot distinguish 5 hmC from 5 methylcytosine 5 mC This kit features a robust and highly specific 5 hmC glucosyltransferase enzyme to specifically tag 5 hmC sites yielding the modified base glucosyl 5 hydroxymethylcytosine g 5 hmC After glucosylation of 5 hmC digestion of DNA with Msp1 allow 5 hmC to be differentiated from 5 mC Msp1 can efficiently digest DNA when a cytosine 5 mC or 5 hmC is present in their recognition site but it is sensitive to the presence of g 5 hmC By exploiting this sensitivity the 5 hmC level of a specific locus can be interrogated by utilizing various downstream applications e g endpoint PCR qPCR Next Generation sequencing etc
    Catalog Number:
    d5411
    Price:
    None
    Applications:
    5-hmC Detection
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research quest 5hmc detection kit
    Purification and identification of EndoG that preferentially cleaves <t>5hmC-modified</t> DNA. Purification scheme, using mouse livers as a starting material, for the endonuclease that preferentially cleaves 5hmC-modified DNA (A). 4.5 ng (2.6 fmol DNA molecules) of either unmodified or 5hmC-modified DNA substrates were incubated with each successive column pool (B). Full column activity fractions are shown in Supplementary Figures S4–S7. 4.5 ng (2.6 fmol DNA molecules) of either cytosine or 5hmC-modified substrates were incubated with 50 ng LiNE from wild-type mice or 50 ng LiNE derived from Endog −/− mice (C). 50 ng LiNE, 6.5 nM Purified EndoG or 6.5 nM Purified EndoG/H128A were incubated with 4.5 ng (2.6 fmol DNA molecules) unmodified or 5hmC-modified 2.7 kbp DNA substrates (D). Quantification of the specific cleavage products resulting from a titration of EndoG and EndoG/H128A (E). S, substrate; P, products.
    The Quest 5 hmC Detection Kit allows for locus specific detection of 5 hydroxymethylcytosine 5 hmC using a simple and efficient reaction setup Traditional methods cannot distinguish 5 hmC from 5 methylcytosine 5 mC This kit features a robust and highly specific 5 hmC glucosyltransferase enzyme to specifically tag 5 hmC sites yielding the modified base glucosyl 5 hydroxymethylcytosine g 5 hmC After glucosylation of 5 hmC digestion of DNA with Msp1 allow 5 hmC to be differentiated from 5 mC Msp1 can efficiently digest DNA when a cytosine 5 mC or 5 hmC is present in their recognition site but it is sensitive to the presence of g 5 hmC By exploiting this sensitivity the 5 hmC level of a specific locus can be interrogated by utilizing various downstream applications e g endpoint PCR qPCR Next Generation sequencing etc
    https://www.bioz.com/result/quest 5hmc detection kit/product/Zymo Research
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    quest 5hmc detection kit - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination"

    Article Title: Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1032

    Purification and identification of EndoG that preferentially cleaves 5hmC-modified DNA. Purification scheme, using mouse livers as a starting material, for the endonuclease that preferentially cleaves 5hmC-modified DNA (A). 4.5 ng (2.6 fmol DNA molecules) of either unmodified or 5hmC-modified DNA substrates were incubated with each successive column pool (B). Full column activity fractions are shown in Supplementary Figures S4–S7. 4.5 ng (2.6 fmol DNA molecules) of either cytosine or 5hmC-modified substrates were incubated with 50 ng LiNE from wild-type mice or 50 ng LiNE derived from Endog −/− mice (C). 50 ng LiNE, 6.5 nM Purified EndoG or 6.5 nM Purified EndoG/H128A were incubated with 4.5 ng (2.6 fmol DNA molecules) unmodified or 5hmC-modified 2.7 kbp DNA substrates (D). Quantification of the specific cleavage products resulting from a titration of EndoG and EndoG/H128A (E). S, substrate; P, products.
    Figure Legend Snippet: Purification and identification of EndoG that preferentially cleaves 5hmC-modified DNA. Purification scheme, using mouse livers as a starting material, for the endonuclease that preferentially cleaves 5hmC-modified DNA (A). 4.5 ng (2.6 fmol DNA molecules) of either unmodified or 5hmC-modified DNA substrates were incubated with each successive column pool (B). Full column activity fractions are shown in Supplementary Figures S4–S7. 4.5 ng (2.6 fmol DNA molecules) of either cytosine or 5hmC-modified substrates were incubated with 50 ng LiNE from wild-type mice or 50 ng LiNE derived from Endog −/− mice (C). 50 ng LiNE, 6.5 nM Purified EndoG or 6.5 nM Purified EndoG/H128A were incubated with 4.5 ng (2.6 fmol DNA molecules) unmodified or 5hmC-modified 2.7 kbp DNA substrates (D). Quantification of the specific cleavage products resulting from a titration of EndoG and EndoG/H128A (E). S, substrate; P, products.

    Techniques Used: Purification, Modification, DNA Purification, Incubation, Activity Assay, Mouse Assay, Derivative Assay, Titration

    EndoG promotes conservative recombination when the DNA is 5hmC-modified. Substrate A lacks the 3′ region of an amplicon containing the EndoG recognition sequence (blue primers) and Substrate B lacks the 5′ region of this same amplicon. If recombination occurs the complete amplicon is restored resulting in a quantifiable product. Recombination is quantified as the recombination amplicon quantity (blue primers) as a percent of the total DNA quantity recovered (orange amplicon). Substrates A and B were either unmodified or 5hmC-modified at every cytosine residues (A). 100 ng of Substrate A and 100 ng of Substrate B were incubated with 15.3 μg LiNE or μg LiNE and 3.2 nM of a specific inhibitor of EndoG (EndoGI) from Drosophila melanogaster as described in the ‘Materials and Methods’ section (B). Data are presented as the mean ± standard deviation, n = 3; statistical significance was assigned using a two-tailed Student's t -test. Recombinant amplicons from the recombination assays in panel B were cloned and sequenced, representative sequences are shown aligned to the expected sequence for a conservative recombinant molecule (top sequence), the relevant sequence of substrate A (sequence second from top) and the relevant sequence of substrate B (sequence third from top) (C). Full sequences are shown in Supplementary Figure S21. Model for the initiation of recombination at 5′-GGGGCCAG-3′ sequences mediated by EndoG and cytosine hydroxymethylation (D).
    Figure Legend Snippet: EndoG promotes conservative recombination when the DNA is 5hmC-modified. Substrate A lacks the 3′ region of an amplicon containing the EndoG recognition sequence (blue primers) and Substrate B lacks the 5′ region of this same amplicon. If recombination occurs the complete amplicon is restored resulting in a quantifiable product. Recombination is quantified as the recombination amplicon quantity (blue primers) as a percent of the total DNA quantity recovered (orange amplicon). Substrates A and B were either unmodified or 5hmC-modified at every cytosine residues (A). 100 ng of Substrate A and 100 ng of Substrate B were incubated with 15.3 μg LiNE or μg LiNE and 3.2 nM of a specific inhibitor of EndoG (EndoGI) from Drosophila melanogaster as described in the ‘Materials and Methods’ section (B). Data are presented as the mean ± standard deviation, n = 3; statistical significance was assigned using a two-tailed Student's t -test. Recombinant amplicons from the recombination assays in panel B were cloned and sequenced, representative sequences are shown aligned to the expected sequence for a conservative recombinant molecule (top sequence), the relevant sequence of substrate A (sequence second from top) and the relevant sequence of substrate B (sequence third from top) (C). Full sequences are shown in Supplementary Figure S21. Model for the initiation of recombination at 5′-GGGGCCAG-3′ sequences mediated by EndoG and cytosine hydroxymethylation (D).

    Techniques Used: Modification, Amplification, Sequencing, Incubation, Standard Deviation, Two Tailed Test, Recombinant, Clone Assay

    A component of LiNE preferentially cleaves 5hmC-modified DNA. 4.5 ng (2.6 fmol DNA molecules) of either cytosine or the fully 5hmC-modified 2.7 kbp substrates (A) were incubated with 25 ng LiNE with an increasing molar excess (0.1- to 10-fold) of poly(dIdC) (B). *, radioactive label; S, substrate; P, products.
    Figure Legend Snippet: A component of LiNE preferentially cleaves 5hmC-modified DNA. 4.5 ng (2.6 fmol DNA molecules) of either cytosine or the fully 5hmC-modified 2.7 kbp substrates (A) were incubated with 25 ng LiNE with an increasing molar excess (0.1- to 10-fold) of poly(dIdC) (B). *, radioactive label; S, substrate; P, products.

    Techniques Used: Modification, Incubation

    The 5hmC-cleavage activity cannot cleave 5meC modified DNA and EndoG preferentially cleaves the sequence 5′-GGGG 5hm CCAG-3′. 4.5 ng each short (130 bp) cytosine, fully 5meC and fully 5hmC substrates (5hmC), 32 P labeled at both ends, were incubated with a titration of LiNE ranging from 37.0 to 333 ng (A). dsOligonucleotides (45 bp) that were singly 5hmC-modified at the indicated position were incubated with purified 12.5 nM EndoG (B). S, substrate; P, products; P 1 , product 1; P 2 , product 2.
    Figure Legend Snippet: The 5hmC-cleavage activity cannot cleave 5meC modified DNA and EndoG preferentially cleaves the sequence 5′-GGGG 5hm CCAG-3′. 4.5 ng each short (130 bp) cytosine, fully 5meC and fully 5hmC substrates (5hmC), 32 P labeled at both ends, were incubated with a titration of LiNE ranging from 37.0 to 333 ng (A). dsOligonucleotides (45 bp) that were singly 5hmC-modified at the indicated position were incubated with purified 12.5 nM EndoG (B). S, substrate; P, products; P 1 , product 1; P 2 , product 2.

    Techniques Used: Activity Assay, Modification, Sequencing, Labeling, Incubation, Titration, Purification

    EndoG both cleaves cytosine hydroxymethylated DNA within the sequence context 5′-GGGGCCAG-3′ and promotes the formation of γ-H2AX foci in vivo . Schematic (not to scale) of the substrate design for the assay, each 4.1 kbp substrate contained either cytosine or 5hmC in place of all the cytosines. The core substrate contains the EndoG recognition sequence while the core mutated substrate has the EndoG recognition sequence mutated (A). 0.5 μg of each of the four DNA substrates was transfected into HeLa cells that were treated with a control or EndoG siRNA 1. The total DNA was recovered 20 h after transfection. Bars on the graph represent the total amount of the core EndoG recognition region remaining (amplicon I) as a fraction of DNA transfected (amplicon II) quantified by qPCR (B). Data are presented as the mean ± standard deviation, n = 7; statistical significance was assigned using a two-tailed Student's t -test. Representative images of γ-H2AX foci and 53BP1 foci observed in HeLa cells stably transfected with either a control plasmid or a plasmid that overexpresses the Tet2 CD. Two different siRNAs targeting EndoG were transfected into both the control and the Tet2 CD cell line and incubated for 48 h (C). γ-H2AX foci were counted and grouped according to the number of foci per cell in each of the six combinations of cell lines evaluated (D).
    Figure Legend Snippet: EndoG both cleaves cytosine hydroxymethylated DNA within the sequence context 5′-GGGGCCAG-3′ and promotes the formation of γ-H2AX foci in vivo . Schematic (not to scale) of the substrate design for the assay, each 4.1 kbp substrate contained either cytosine or 5hmC in place of all the cytosines. The core substrate contains the EndoG recognition sequence while the core mutated substrate has the EndoG recognition sequence mutated (A). 0.5 μg of each of the four DNA substrates was transfected into HeLa cells that were treated with a control or EndoG siRNA 1. The total DNA was recovered 20 h after transfection. Bars on the graph represent the total amount of the core EndoG recognition region remaining (amplicon I) as a fraction of DNA transfected (amplicon II) quantified by qPCR (B). Data are presented as the mean ± standard deviation, n = 7; statistical significance was assigned using a two-tailed Student's t -test. Representative images of γ-H2AX foci and 53BP1 foci observed in HeLa cells stably transfected with either a control plasmid or a plasmid that overexpresses the Tet2 CD. Two different siRNAs targeting EndoG were transfected into both the control and the Tet2 CD cell line and incubated for 48 h (C). γ-H2AX foci were counted and grouped according to the number of foci per cell in each of the six combinations of cell lines evaluated (D).

    Techniques Used: Sequencing, In Vivo, Transfection, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test, Stable Transfection, Plasmid Preparation, Incubation

    2) Product Images from "Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system"

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

    Journal: PeerJ

    doi: 10.7717/peerj.230

    Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.
    Figure Legend Snippet: Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.

    Techniques Used: Sequencing, Triple Knockout, Mutagenesis, Clone Assay, Purification, Concentration Assay

    3) Product Images from "5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes"

    Article Title: 5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-5-r46

    5hmC deposition precedes active DNA demethylation in human monocytes . ( A ) Positions of regions (purple) measured by MALDI-TOF analysis of bisulfite converted DNA (MassARRAY, in B ) and of primers (red) used for hMeDIP qPCR (in C ) are shown relative to positions of CpG dinucleotides (green) and neighboring genes (blue). Tracks were generated using the UCSC Genome Browser. (B) MassARRAY analysis of bisulfite-converted DNA at four loci that show active DNA demethylation during monocyte to DC differentiation, as well as for two control regions (values are mean of n ≥4). Data are presented as heatmaps. The methylation content (including both 5mC and 5hmC) is indicated by coloring (yellow: no methylation, dark blue: 100% methylation) with each box representing a single CpG dinucleotide and each row representing the succession of CpGs measured. Grey boxes indicate CpGs that were not detected by MALDI-TOF MS. Red asterisks mark the CpGs that are shown in (C). Methylation ratios of single CpG units for individual donors are also provided in Table S2 in Additional File 2 . (C) Dynamics of DNA methylation (5mC + 5hmC) and hydroxymethylation (5hmC) during monocytic differentiation. DNA methylation levels of single CpGs as measured by MassARRAY (open squares) are compared with 5hmC enrichment (measured by hMeDIP, red squares) at the same loci shown in (B) ( n ≥4, values are mean + or - SD). Exact genomic positions of analyzed CpG residues are given in Table S3 in Additional File 3 .
    Figure Legend Snippet: 5hmC deposition precedes active DNA demethylation in human monocytes . ( A ) Positions of regions (purple) measured by MALDI-TOF analysis of bisulfite converted DNA (MassARRAY, in B ) and of primers (red) used for hMeDIP qPCR (in C ) are shown relative to positions of CpG dinucleotides (green) and neighboring genes (blue). Tracks were generated using the UCSC Genome Browser. (B) MassARRAY analysis of bisulfite-converted DNA at four loci that show active DNA demethylation during monocyte to DC differentiation, as well as for two control regions (values are mean of n ≥4). Data are presented as heatmaps. The methylation content (including both 5mC and 5hmC) is indicated by coloring (yellow: no methylation, dark blue: 100% methylation) with each box representing a single CpG dinucleotide and each row representing the succession of CpGs measured. Grey boxes indicate CpGs that were not detected by MALDI-TOF MS. Red asterisks mark the CpGs that are shown in (C). Methylation ratios of single CpG units for individual donors are also provided in Table S2 in Additional File 2 . (C) Dynamics of DNA methylation (5mC + 5hmC) and hydroxymethylation (5hmC) during monocytic differentiation. DNA methylation levels of single CpGs as measured by MassARRAY (open squares) are compared with 5hmC enrichment (measured by hMeDIP, red squares) at the same loci shown in (B) ( n ≥4, values are mean + or - SD). Exact genomic positions of analyzed CpG residues are given in Table S3 in Additional File 3 .

    Techniques Used: Real-time Polymerase Chain Reaction, Generated, Methylation, Mass Spectrometry, DNA Methylation Assay

    4) Product Images from "TET family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma"

    Article Title: TET family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma

    Journal: Oncotarget

    doi:

    A. Kaplan–Meier curves for overall survival according to 5 hmC status in ESCCs. B. Kaplan–Meier curves for overall survival according to TET2 stutus in ESCCs.
    Figure Legend Snippet: A. Kaplan–Meier curves for overall survival according to 5 hmC status in ESCCs. B. Kaplan–Meier curves for overall survival according to TET2 stutus in ESCCs.

    Techniques Used:

    Correlation between 5-hmC expression and mRNA levels of TETs family in ESCC A. TET1 expression. B. TET2 expression. C. TET3 expression.
    Figure Legend Snippet: Correlation between 5-hmC expression and mRNA levels of TETs family in ESCC A. TET1 expression. B. TET2 expression. C. TET3 expression.

    Techniques Used: Expressing

    Correlation between the LINE-1 methylation levels and 5-hmC expression or mRNA levels of TETs family A. Pyrosequencing for LINE-1 methylation level. B. Relationship between LINE-1 methylation level and 5 hmC level. ( n = 89, P = 0.0002, r = 0.39). C. Relationship between LINE-1 methylation level and TET1 expression. D. Relationship between LINE-1 methylation level and TET2 expression. E. Relationship between LINE-1 methylation level and TET3 expression.
    Figure Legend Snippet: Correlation between the LINE-1 methylation levels and 5-hmC expression or mRNA levels of TETs family A. Pyrosequencing for LINE-1 methylation level. B. Relationship between LINE-1 methylation level and 5 hmC level. ( n = 89, P = 0.0002, r = 0.39). C. Relationship between LINE-1 methylation level and TET1 expression. D. Relationship between LINE-1 methylation level and TET2 expression. E. Relationship between LINE-1 methylation level and TET3 expression.

    Techniques Used: Methylation, Expressing

    Model for the relationship between 5-hmC and TET2 in ESCC
    Figure Legend Snippet: Model for the relationship between 5-hmC and TET2 in ESCC

    Techniques Used:

    5-hmC expression in ESCC A. ELISA assay shows percentages of 5-hmC levels in 33 matched ESCC normal mucosa specimens. B. Immunohistochemical staining for 5-hmC in normal esophageal epithelium and ESCC.
    Figure Legend Snippet: 5-hmC expression in ESCC A. ELISA assay shows percentages of 5-hmC levels in 33 matched ESCC normal mucosa specimens. B. Immunohistochemical staining for 5-hmC in normal esophageal epithelium and ESCC.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

    5) Product Images from "Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2"

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2

    Journal: Translational Oncology

    doi:

    Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained
    Figure Legend Snippet: Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction

    6) Product Images from "AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation"

    Article Title: AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.17

    AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.
    Figure Legend Snippet: AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, shRNA, Immunoprecipitation, Methylation, Activation Assay

    5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value
    Figure Legend Snippet: 5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value

    Techniques Used: Labeling, Staining, Real-time Polymerase Chain Reaction, Genome Wide, Methylated DNA Immunoprecipitation

    7) Product Images from "AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation"

    Article Title: AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.17

    AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.
    Figure Legend Snippet: AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, shRNA, Immunoprecipitation, Methylation, Activation Assay

    5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value
    Figure Legend Snippet: 5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value

    Techniques Used: Labeling, Staining, Real-time Polymerase Chain Reaction, Genome Wide, Methylated DNA Immunoprecipitation

    8) Product Images from "5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes"

    Article Title: 5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

    Journal: Genome Biology

    doi: 10.1186/gb-2013-14-5-r46

    5hmC deposition precedes active DNA demethylation in human monocytes . ( A ) Positions of regions (purple) measured by MALDI-TOF analysis of bisulfite converted DNA (MassARRAY, in B ) and of primers (red) used for hMeDIP qPCR (in C ) are shown relative to positions of CpG dinucleotides (green) and neighboring genes (blue). Tracks were generated using the UCSC Genome Browser. (B) MassARRAY analysis of bisulfite-converted DNA at four loci that show active DNA demethylation during monocyte to DC differentiation, as well as for two control regions (values are mean of n ≥4). Data are presented as heatmaps. The methylation content (including both 5mC and 5hmC) is indicated by coloring (yellow: no methylation, dark blue: 100% methylation) with each box representing a single CpG dinucleotide and each row representing the succession of CpGs measured. Grey boxes indicate CpGs that were not detected by MALDI-TOF MS. Red asterisks mark the CpGs that are shown in (C). Methylation ratios of single CpG units for individual donors are also provided in Table S2 in Additional File 2 . (C) Dynamics of DNA methylation (5mC + 5hmC) and hydroxymethylation (5hmC) during monocytic differentiation. DNA methylation levels of single CpGs as measured by MassARRAY (open squares) are compared with 5hmC enrichment (measured by hMeDIP, red squares) at the same loci shown in (B) ( n ≥4, values are mean + or - SD). Exact genomic positions of analyzed CpG residues are given in Table S3 in Additional File 3 .
    Figure Legend Snippet: 5hmC deposition precedes active DNA demethylation in human monocytes . ( A ) Positions of regions (purple) measured by MALDI-TOF analysis of bisulfite converted DNA (MassARRAY, in B ) and of primers (red) used for hMeDIP qPCR (in C ) are shown relative to positions of CpG dinucleotides (green) and neighboring genes (blue). Tracks were generated using the UCSC Genome Browser. (B) MassARRAY analysis of bisulfite-converted DNA at four loci that show active DNA demethylation during monocyte to DC differentiation, as well as for two control regions (values are mean of n ≥4). Data are presented as heatmaps. The methylation content (including both 5mC and 5hmC) is indicated by coloring (yellow: no methylation, dark blue: 100% methylation) with each box representing a single CpG dinucleotide and each row representing the succession of CpGs measured. Grey boxes indicate CpGs that were not detected by MALDI-TOF MS. Red asterisks mark the CpGs that are shown in (C). Methylation ratios of single CpG units for individual donors are also provided in Table S2 in Additional File 2 . (C) Dynamics of DNA methylation (5mC + 5hmC) and hydroxymethylation (5hmC) during monocytic differentiation. DNA methylation levels of single CpGs as measured by MassARRAY (open squares) are compared with 5hmC enrichment (measured by hMeDIP, red squares) at the same loci shown in (B) ( n ≥4, values are mean + or - SD). Exact genomic positions of analyzed CpG residues are given in Table S3 in Additional File 3 .

    Techniques Used: Real-time Polymerase Chain Reaction, Generated, Methylation, Mass Spectrometry, DNA Methylation Assay

    9) Product Images from "Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system"

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

    Journal: PeerJ

    doi: 10.7717/peerj.230

    Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.
    Figure Legend Snippet: Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.

    Techniques Used: Sequencing, Triple Knockout, Mutagenesis, Clone Assay, Purification, Concentration Assay

    10) Product Images from "Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system"

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

    Journal: PeerJ

    doi: 10.7717/peerj.230

    Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.
    Figure Legend Snippet: Simultaneous targeting of the Tet1, Tet2 and Tet3 genes in mouse haploid ESCs. (A) Sequencing of Tet1 , Tet2 and Tet3 triple knockout mutant clones. The PAM sequences are shown in red and the boxed areas indicate the restriction sites in the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions. (B, C) Quantification of the 5hmC content in triple knockout clones. Analysis of the global 5hmC levels (B) and the 5hmC levels in the Ecat1 gene (C) in DNA from two triple knockout (TKO) haploid ESC clones. The quantification of genomic 5hmC was based on the specific transfer of radiolabeled glucose to 5hmC by a purified glucosyltransferase. Data are represented as the mean + SD of n = 3 replicate measurements and are shown as a percentage of the 5hmC levels in the wild type (WT). (D) Sequencing of Tet1 , Tet2, and Tet3 triple knockout mutant clones was obtained following treatment with a high concentration of puromycin. PAM sequences are shown in red and the boxed areas indicate the restriction sites of the target regions. Lower case letters indicate insertion mutations and arrows indicate the sites of insertions.

    Techniques Used: Sequencing, Triple Knockout, Mutagenesis, Clone Assay, Purification, Concentration Assay

    11) Product Images from "Cytosine 5-hydroxymethylation regulates VHL gene expression in renal clear cell carcinoma"

    Article Title: Cytosine 5-hydroxymethylation regulates VHL gene expression in renal clear cell carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19070

    The structure of the VHL gene and its detected 5hmC sites
    Figure Legend Snippet: The structure of the VHL gene and its detected 5hmC sites

    Techniques Used:

    VHL 5hmC analysis of normal, RCC tissues and different RCC grades RCC and normal indicate renal clear cell carcinoma and matched adjacent benign tissues. S1, S2, S3 and S4 indicate the four Msp I sites (CCGG) used for 5hmC analysis. G indicates the pathological grade of RCC. * P
    Figure Legend Snippet: VHL 5hmC analysis of normal, RCC tissues and different RCC grades RCC and normal indicate renal clear cell carcinoma and matched adjacent benign tissues. S1, S2, S3 and S4 indicate the four Msp I sites (CCGG) used for 5hmC analysis. G indicates the pathological grade of RCC. * P

    Techniques Used:

    12) Product Images from "Targets and genomic constraints of ectopic Dnmt3b expression"

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression

    Journal: eLife

    doi: 10.7554/eLife.40757

    Dnmt3b overexpression in human cancer types and an ectopic mouse model. ( A ) Incidence of DNMT3A and DNMT3B mutation and overexpression in human cancers, based on data from The Cancer Genome Atlas (TCGA). H/N = head and neck. ( B ) Fraction of hypermethylated (defined as ≥0.3 increase in average CpG methylation) CpG islands (CGIs) in liver hepatocellular carcinomas and colorectal adenocarcinoma. Each line represents a pair of matched normal and tumor samples from a single patient. Patients that show overexpression of DNMT3A or DNMT3B ( z -score ≥ 2) in the tumor sample are shown in dark green. ( C ) Mean CGI methylation vs percentage of hypermethylated CGIs (methylation ≥ 0.3) for 11 tissues harvested from age-matched control and Dnmt3b overexpression (3b OE) mice (0 – 1.5M dox). Data are based on RRBS profiles for matched CGIs ( n = 7,467). ( D ) Heatmap of methylation levels at differentially methylated CGIs (FDR q -value
    Figure Legend Snippet: Dnmt3b overexpression in human cancer types and an ectopic mouse model. ( A ) Incidence of DNMT3A and DNMT3B mutation and overexpression in human cancers, based on data from The Cancer Genome Atlas (TCGA). H/N = head and neck. ( B ) Fraction of hypermethylated (defined as ≥0.3 increase in average CpG methylation) CpG islands (CGIs) in liver hepatocellular carcinomas and colorectal adenocarcinoma. Each line represents a pair of matched normal and tumor samples from a single patient. Patients that show overexpression of DNMT3A or DNMT3B ( z -score ≥ 2) in the tumor sample are shown in dark green. ( C ) Mean CGI methylation vs percentage of hypermethylated CGIs (methylation ≥ 0.3) for 11 tissues harvested from age-matched control and Dnmt3b overexpression (3b OE) mice (0 – 1.5M dox). Data are based on RRBS profiles for matched CGIs ( n = 7,467). ( D ) Heatmap of methylation levels at differentially methylated CGIs (FDR q -value

    Techniques Used: Over Expression, Mutagenesis, CpG Methylation Assay, Methylation, Mouse Assay

    13) Product Images from "Evaluation of Post-Mortem Effects on Global Brain DNA Methylation and Hydroxymethylation"

    Article Title: Evaluation of Post-Mortem Effects on Global Brain DNA Methylation and Hydroxymethylation

    Journal: Basic & clinical pharmacology & toxicology

    doi: 10.1111/bcpt.12875

    Effects of post-mortem interval and tissue heat stabilization (HS) on global DNA methylation in cerebellum measured by LUMA (A) and ELISA (B). To study the effect of post-mortem interval, the cerebellum from three groups of neonatal and adult animals were kept at room temperature for 0–540 min. before they were snap-frozen (SF) and stored at −80°C. To determine the effect of HS, the cerebellum from three groups of neonatal animals were instantly heat-stabilized and kept at room temperature for 0–540 min. before storage. Values represent mean ± S.E.M. * p
    Figure Legend Snippet: Effects of post-mortem interval and tissue heat stabilization (HS) on global DNA methylation in cerebellum measured by LUMA (A) and ELISA (B). To study the effect of post-mortem interval, the cerebellum from three groups of neonatal and adult animals were kept at room temperature for 0–540 min. before they were snap-frozen (SF) and stored at −80°C. To determine the effect of HS, the cerebellum from three groups of neonatal animals were instantly heat-stabilized and kept at room temperature for 0–540 min. before storage. Values represent mean ± S.E.M. * p

    Techniques Used: DNA Methylation Assay, Enzyme-linked Immunosorbent Assay

    14) Product Images from "TET2 is a Master Regulator of Smooth Muscle Cell Plasticity"

    Article Title: TET2 is a Master Regulator of Smooth Muscle Cell Plasticity

    Journal: Circulation

    doi: 10.1161/CIRCULATIONAHA.113.002887

    TET2 modulation alters 5-hmC levels and the intimal hyperplastic response to arterial injury. (A) EVG staining 3 weeks post-injury of femoral arteries transduced with either a control, TET2 knockdown or TET2 overexpressing virus. (B) Quantitation of the
    Figure Legend Snippet: TET2 modulation alters 5-hmC levels and the intimal hyperplastic response to arterial injury. (A) EVG staining 3 weeks post-injury of femoral arteries transduced with either a control, TET2 knockdown or TET2 overexpressing virus. (B) Quantitation of the

    Techniques Used: Staining, Transduction, Quantitation Assay

    TET2 is downregulated during vascular injury. (A) TET2 and 5-hmC immunostaining of cross-sections from uninjured contralateral control mouse femoral arteries (top row) or injured femoral arteries (bottom row) collected 3 weeks following wire injury. Nuclei
    Figure Legend Snippet: TET2 is downregulated during vascular injury. (A) TET2 and 5-hmC immunostaining of cross-sections from uninjured contralateral control mouse femoral arteries (top row) or injured femoral arteries (bottom row) collected 3 weeks following wire injury. Nuclei

    Techniques Used: Immunostaining

    TET2 and 5-hmC levels are high in mature human SMC and are lost in atherosclerotic lesions. (A) H E staining (row 1), TET2 and MYH11 immunostaining (rows 2–5) of human coronary arteries with various degrees of atherosclerosis. The boxed
    Figure Legend Snippet: TET2 and 5-hmC levels are high in mature human SMC and are lost in atherosclerotic lesions. (A) H E staining (row 1), TET2 and MYH11 immunostaining (rows 2–5) of human coronary arteries with various degrees of atherosclerosis. The boxed

    Techniques Used: Staining, Immunostaining

    TET2 regulates SMC phenotype. (A) Western blot of contractile genes following TET2 knockdown (shTET2) in hCASMC. (B) Dot blot of 5-hmC in control (shCTRL) compared to shTET2 cells. (C–D) qPCR for contractile genes in shCTRL or shTET2 hCASMC treated
    Figure Legend Snippet: TET2 regulates SMC phenotype. (A) Western blot of contractile genes following TET2 knockdown (shTET2) in hCASMC. (B) Dot blot of 5-hmC in control (shCTRL) compared to shTET2 cells. (C–D) qPCR for contractile genes in shCTRL or shTET2 hCASMC treated

    Techniques Used: Western Blot, Dot Blot, Real-time Polymerase Chain Reaction

    TET2 binds to SMC promoters to regulate 5-hmC and modify histones at SMC loci. (A) ChIP assay demonstrating TET2 enrichment at SRF , MYOCD and MYH11 promoters during SMC differentiation. hCASMC were treated with rapamycin or PDGF-BB for 24 hours. Data
    Figure Legend Snippet: TET2 binds to SMC promoters to regulate 5-hmC and modify histones at SMC loci. (A) ChIP assay demonstrating TET2 enrichment at SRF , MYOCD and MYH11 promoters during SMC differentiation. hCASMC were treated with rapamycin or PDGF-BB for 24 hours. Data

    Techniques Used: Chromatin Immunoprecipitation

    15) Product Images from "Epigenetic Modifications in the Biology of Nonalcoholic Fatty Liver Disease"

    Article Title: Epigenetic Modifications in the Biology of Nonalcoholic Fatty Liver Disease

    Journal: Medicine

    doi: 10.1097/MD.0000000000001480

    Global levels of 5-hydroxymethylcytosine (5-hmC) in the liver tissue of patients with NAFLD and near normal liver histology (NNLH). A: Correlation between log-transformed liver mtDNA/nDNA ratio and log-transformed global liver 5-hmC levels measured by a sandwich-based enzyme-linked immunosorbent assay employing an anti-5-hydroxymethylcytosine polyclonal antibody. B and C: A representative specimen of immunostaining for 5-hmC in the liver of a patient with NNLH and NAFLD, respectively. Arrows indicate nonnuclear areas of 5-hmC staining. D: Magnification shows 5-hmC preferentially localized at the edge of lipid-laden hepatocytes; N = nucleus. Representative electron micrograph of hepatocyte isolated from an NASH patient showing a lipid droplet (LD) and mitochondria (M) in close physical association with LDs. Magnification: ×3000. 5-hmC immunoreactivity was examined using light microscopy of liver sections, while counterstaining was performed with eosin. Original magnification: ×400. NAFLD = nonalcoholic fatty liver disease.
    Figure Legend Snippet: Global levels of 5-hydroxymethylcytosine (5-hmC) in the liver tissue of patients with NAFLD and near normal liver histology (NNLH). A: Correlation between log-transformed liver mtDNA/nDNA ratio and log-transformed global liver 5-hmC levels measured by a sandwich-based enzyme-linked immunosorbent assay employing an anti-5-hydroxymethylcytosine polyclonal antibody. B and C: A representative specimen of immunostaining for 5-hmC in the liver of a patient with NNLH and NAFLD, respectively. Arrows indicate nonnuclear areas of 5-hmC staining. D: Magnification shows 5-hmC preferentially localized at the edge of lipid-laden hepatocytes; N = nucleus. Representative electron micrograph of hepatocyte isolated from an NASH patient showing a lipid droplet (LD) and mitochondria (M) in close physical association with LDs. Magnification: ×3000. 5-hmC immunoreactivity was examined using light microscopy of liver sections, while counterstaining was performed with eosin. Original magnification: ×400. NAFLD = nonalcoholic fatty liver disease.

    Techniques Used: Transformation Assay, Sandwich ELISA, Immunostaining, Staining, Isolation, Light Microscopy

    16) Product Images from "AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation"

    Article Title: AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.17

    AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.
    Figure Legend Snippet: AF9 and TET2 cooperate to regulate the conversion of 5mC to 5hmC at neural gene loci. ( a ) 5hmC-positive cells were co-lobeled with TUJ1 in AF9-induced neurons. Control (Ctrl) or AF9-overexpressing (AF9-IRES-RFP) cells were differentiated for 12 days according to the protocol shown in Figure 2A and co-stained with anti-TUJ1 and anti-5hmC antibodies. Bar, 100 μm. ( b ) Glu-qPCR analysis of 5hmC levels in day 8 or day 12 differentiated cells as indicated. Ctrl/D8, day 8 cells expressing control vector; AF9 OV/D8, day 8 cells expressing AF9-IRES-RFP; Ctrl KD/D12, day 12 cells expressing control shRNA; AF9 KD1/D12 or AF9 KD2/D12, day 12 cells expressing AF9 shRNA1 or shRNA2; Ctrl KD'/D12, day 12 cells expressing control shRNA; TET2 KD1/D12 or TET2 KD2/D12, day 12 cells expressing TET2 shRNA1 or shRNA2; AF9OV+Ctrl KD’/D8, day 8 cells expressing control shRNA and AF9-IRES-RFP; AF9OV+TET2 KD1/D8 or AF9OV+TET2 KD2/D8, day 8 cells expressing TET2 shRNA1 or shRNA2 and AF9-IRES-RFP. ( c ) DNA immunoprecipitation was performed using an anti-5mC antibody. DIP-qPCR analysis was conducted to determine the 5mC levels relative to input DNA in the same group of cells in ( b ). ( d ) A model for the role of the AF9–TET2 complex in the modulation of human embryonic stem cell (hESC) neural differentiation. In hESCs, some neural gene loci are methylated and inactive. During neural differentiation, upregulated AF9 are enriched in the neurodevelopmental gene loci by recognizing the AAC-containing elements. Then, AF9 recruits TET2 to nearby C-rich DNA sequences through the TET2 catalytic domain, and the AF9–TET2 complex cooperates to direct the 5mC conversion to 5hmC or to unmethylated cytosine ( c ). The AF9–TET2 complex-mediated 5mC-to-5hmC conversions results in the activation of multiple neural genes and hESC differentiation into neurons.

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, shRNA, Immunoprecipitation, Methylation, Activation Assay

    5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value
    Figure Legend Snippet: 5mC conversion to 5hmC at neural gene loci during hESC neural differentiation. ( a ) The co-labeling of 5hmC with TUJ1, TET2, and AF9 in human neural cells. Cells were stained for 5hmC and co-stained for TUJ1, TET2, or AF9 in day 22 cells. Bars, 50 μm. ( b ) Glu-qPCR showing quantitative levels of 5hmC at MSP1 (CCGG) sites in the co-occupied regions of neural-related gene loci from day 0 to day 16. R1-R3 are shown as in Figure 5c . ( c ) Genome-wide distributions of 5mC and 5hmC were determined by MeDIP-seq and hMeDIP-seq. The peak distances to transcriptional start sites (TSSs) are shown. The genes that were enriched with peaks within 5 kb upstream or downstream of TSSs are defined as 5mC or 5hmC possibly regulated genes. The numbers of overlapped 5mC- and 5hmC- enriched genes ( P -value

    Techniques Used: Labeling, Staining, Real-time Polymerase Chain Reaction, Genome Wide, Methylated DNA Immunoprecipitation

    17) Product Images from "Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways"

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006987

    Gene expression and 5hmC levels are abnormal in tet2 -/- ;tet3 -/- mutant eyes at 72hpf. (A-B) GO analysis for biological pathways was performed using DAVID. Numbers in parentheses indicate number of genes enriched in each GO term. P-value cutoff = 0.001. In situ hybridization of (C-E) medium-wave sensitive ( opn1mw1 ) and (F-H) short-wave sensitive opsin ( opn1sw1 ). Transcripts of both genes are only detected in a few cells of the ventral retina in tet2 -/- ;tet3 -/- embryos (arrows in E,H; n > 8). natriuretic peptide a ( nppa ) is normally expressed in the heart (I), and is not detected in the retina of wild-type embryos (J; n > 8). (K) In tet2 -/- ;tet3 -/- embryos, ectopic nppa expressing cells were detected throughout the retina and brain (arrows) of all embryos examined (n = 16/16). (L) Bisulfite sequencing did not identify any changes in DNA methylation in any of the sixteen RNA-seq-identified target loci examined ( S4 Table ), including the nppa gene body. Bisulfite reads covering part of the first intron and second exon of nppa gene body are shown as black (methylated) or white (unmethylated). (M) Site-specific 5hmC quantification detected a significant, 20-fold reduction in 5hmC levels in the nppa gene body of 72hpf tet2 -/- ;tet3 -/- embryonic eye tissue, when compared to phenotypically wild-type siblings (p = 0.0038; two-tailed, unpaired t-test).
    Figure Legend Snippet: Gene expression and 5hmC levels are abnormal in tet2 -/- ;tet3 -/- mutant eyes at 72hpf. (A-B) GO analysis for biological pathways was performed using DAVID. Numbers in parentheses indicate number of genes enriched in each GO term. P-value cutoff = 0.001. In situ hybridization of (C-E) medium-wave sensitive ( opn1mw1 ) and (F-H) short-wave sensitive opsin ( opn1sw1 ). Transcripts of both genes are only detected in a few cells of the ventral retina in tet2 -/- ;tet3 -/- embryos (arrows in E,H; n > 8). natriuretic peptide a ( nppa ) is normally expressed in the heart (I), and is not detected in the retina of wild-type embryos (J; n > 8). (K) In tet2 -/- ;tet3 -/- embryos, ectopic nppa expressing cells were detected throughout the retina and brain (arrows) of all embryos examined (n = 16/16). (L) Bisulfite sequencing did not identify any changes in DNA methylation in any of the sixteen RNA-seq-identified target loci examined ( S4 Table ), including the nppa gene body. Bisulfite reads covering part of the first intron and second exon of nppa gene body are shown as black (methylated) or white (unmethylated). (M) Site-specific 5hmC quantification detected a significant, 20-fold reduction in 5hmC levels in the nppa gene body of 72hpf tet2 -/- ;tet3 -/- embryonic eye tissue, when compared to phenotypically wild-type siblings (p = 0.0038; two-tailed, unpaired t-test).

    Techniques Used: Expressing, Mutagenesis, In Situ Hybridization, Methylation Sequencing, DNA Methylation Assay, RNA Sequencing Assay, Methylation, Two Tailed Test

    tet2 -/- ;tet3 -/- mutants are deficient in 5mC → 5hmC conversion and display abnormalities in retinal development. (A) tet2 au59 mutants possess a 10bp deletion resulting in a frameshift, insertion of 37 incorrect aa’s (red), and a premature stop codon, truncating the protein at amino acid (aa) 444 of 1,716aa. (B) tet3 au60 mutants possess a 22bp deletion, resulting in a frameshift, insertion of 92 incorrect aa’s (red), and a premature stop codon, truncating the protein at aa 1,304 of 2,052. (C) Genomic DNA isolated from 2dpf tet2 -/- ;tet3 -/- mutants shows a > 9 fold reduction in global 5hmC levels when compared to phenotypically wild-type siblings and genetically wild-type embryos in an ELISA (n = 20 embryos, p = 0.004 and p = 0.0005, respectively; Error bars = ± 1 S.D.). (D) At 36hpf, tet2 -/- ;tet3 -/- mutants are identifiable based on a kinked head and slightly enlarged brain (arrow). (E,F) Retinae of 36hpf tet2 -/- ;tet3 -/- mutants are morphologically similar to wild-types. (G) At 2dpf, tet2 -/- ;tet3 -/- mutants are microphthalmic, possess cardiac edema and their heads are smaller than phenotypically wildtype siblings. (H-I) The retina is not laminated, with retinal cells appearing progenitor-like in morphology when compared to phenotypically wild-type siblings. (J) Cardiac edema becomes progressively enlarged at 3dpf in tet2 -/- ; tet3 -/- embryos. (K,L) The retina remains poorly laminated in tet2 -/- ;tet3 -/- mutants, and they lack a morphologically obvious optic nerve (arrowhead in sibling). (M) A 4dpf tet2 -/- ; tet3 -/- embryos do not possess an inflated swim bladder (arrow). (N,O) The retina remains poorly laminated and they lack a morphologically obvious optic nerve (arrowhead in sibling). DNA (green), F-actin (cyan). Dorsal is up and anterior to the left. Scale bar = 80μm.
    Figure Legend Snippet: tet2 -/- ;tet3 -/- mutants are deficient in 5mC → 5hmC conversion and display abnormalities in retinal development. (A) tet2 au59 mutants possess a 10bp deletion resulting in a frameshift, insertion of 37 incorrect aa’s (red), and a premature stop codon, truncating the protein at amino acid (aa) 444 of 1,716aa. (B) tet3 au60 mutants possess a 22bp deletion, resulting in a frameshift, insertion of 92 incorrect aa’s (red), and a premature stop codon, truncating the protein at aa 1,304 of 2,052. (C) Genomic DNA isolated from 2dpf tet2 -/- ;tet3 -/- mutants shows a > 9 fold reduction in global 5hmC levels when compared to phenotypically wild-type siblings and genetically wild-type embryos in an ELISA (n = 20 embryos, p = 0.004 and p = 0.0005, respectively; Error bars = ± 1 S.D.). (D) At 36hpf, tet2 -/- ;tet3 -/- mutants are identifiable based on a kinked head and slightly enlarged brain (arrow). (E,F) Retinae of 36hpf tet2 -/- ;tet3 -/- mutants are morphologically similar to wild-types. (G) At 2dpf, tet2 -/- ;tet3 -/- mutants are microphthalmic, possess cardiac edema and their heads are smaller than phenotypically wildtype siblings. (H-I) The retina is not laminated, with retinal cells appearing progenitor-like in morphology when compared to phenotypically wild-type siblings. (J) Cardiac edema becomes progressively enlarged at 3dpf in tet2 -/- ; tet3 -/- embryos. (K,L) The retina remains poorly laminated in tet2 -/- ;tet3 -/- mutants, and they lack a morphologically obvious optic nerve (arrowhead in sibling). (M) A 4dpf tet2 -/- ; tet3 -/- embryos do not possess an inflated swim bladder (arrow). (N,O) The retina remains poorly laminated and they lack a morphologically obvious optic nerve (arrowhead in sibling). DNA (green), F-actin (cyan). Dorsal is up and anterior to the left. Scale bar = 80μm.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2"

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2

    Journal: Translational Oncology

    doi:

    (A) Decreased mRNA expression of LZTS1 and TET1 in patients with breast cancer and their correlation to 5hmC levels. Upper panel reflects mRNA expression of LZTS1 and TET1. Δ C t method was applied on qPCR data followed by division to gain a straightforward
    Figure Legend Snippet: (A) Decreased mRNA expression of LZTS1 and TET1 in patients with breast cancer and their correlation to 5hmC levels. Upper panel reflects mRNA expression of LZTS1 and TET1. Δ C t method was applied on qPCR data followed by division to gain a straightforward

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained
    Figure Legend Snippet: Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction

    19) Product Images from "DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria"

    Article Title: DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1012311108

    mtDNA contains both 5mC and 5hmC. ( A ) mtDNA immunoprecipitation using antibody directed against 5mC shows 10- to 20-fold enrichment compared with IgG controls. ( B ) mtDNA immunoprecipitation using antibody directed against 5hmC demonstrates substantial levels of this modified base in mtDNA. ( C ) Site-specific detection of 5hmC in mtDNA. DNA was incubated in the presence or absence of β-gt, fully methylated with M.Sss 1 and M.CviP 1, and cleaved with Gla1. The degree of protection from cleavage was assessed using qPCR with mtDNA-specific primers. Data shown represent mean ± SD of six replicates from two independent experiments, normalized to input DNA.
    Figure Legend Snippet: mtDNA contains both 5mC and 5hmC. ( A ) mtDNA immunoprecipitation using antibody directed against 5mC shows 10- to 20-fold enrichment compared with IgG controls. ( B ) mtDNA immunoprecipitation using antibody directed against 5hmC demonstrates substantial levels of this modified base in mtDNA. ( C ) Site-specific detection of 5hmC in mtDNA. DNA was incubated in the presence or absence of β-gt, fully methylated with M.Sss 1 and M.CviP 1, and cleaved with Gla1. The degree of protection from cleavage was assessed using qPCR with mtDNA-specific primers. Data shown represent mean ± SD of six replicates from two independent experiments, normalized to input DNA.

    Techniques Used: Immunoprecipitation, Modification, Incubation, Methylation, Real-time Polymerase Chain Reaction

    20) Product Images from "Activation of the ESC pluripotency factor OCT4 in smooth muscle cells is atheroprotective"

    Article Title: Activation of the ESC pluripotency factor OCT4 in smooth muscle cells is atheroprotective

    Journal: Nature medicine

    doi: 10.1038/nm.4109

    Activation of the Oct4 promoter in vitro and in vivo was associated with increased hydroxymethylation ( a ) Bisulfite sequencing analysis of the Oct4 promoter in cultured SMCs in response to hypoxia ± POVPC versus normoxia ± POVPC. See Supplementary Fig. 14 for more details. Open circles indicate unmethylated cytosines, and closed circles indicate methylated cytosines. ( b ) Sequence specific detection of 5-hmC at the Oct4 promoter within the aortic arch regions of Apoe −/− mice fed a high-fat diet as compared to the control age-matched ApoE +/+ mice fed a chow diet as determined by glycosylation-coupled methylation sensitive qPCR. ** P
    Figure Legend Snippet: Activation of the Oct4 promoter in vitro and in vivo was associated with increased hydroxymethylation ( a ) Bisulfite sequencing analysis of the Oct4 promoter in cultured SMCs in response to hypoxia ± POVPC versus normoxia ± POVPC. See Supplementary Fig. 14 for more details. Open circles indicate unmethylated cytosines, and closed circles indicate methylated cytosines. ( b ) Sequence specific detection of 5-hmC at the Oct4 promoter within the aortic arch regions of Apoe −/− mice fed a high-fat diet as compared to the control age-matched ApoE +/+ mice fed a chow diet as determined by glycosylation-coupled methylation sensitive qPCR. ** P

    Techniques Used: Activation Assay, In Vitro, In Vivo, Methylation Sequencing, Cell Culture, Methylation, Sequencing, Mouse Assay, Real-time Polymerase Chain Reaction

    21) Product Images from "Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2"

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2

    Journal: Translational Oncology

    doi:

    (A) Decreased mRNA expression of LZTS1 and TET1 in patients with breast cancer and their correlation to 5hmC levels. Upper panel reflects mRNA expression of LZTS1 and TET1. Δ C t method was applied on qPCR data followed by division to gain a straightforward
    Figure Legend Snippet: (A) Decreased mRNA expression of LZTS1 and TET1 in patients with breast cancer and their correlation to 5hmC levels. Upper panel reflects mRNA expression of LZTS1 and TET1. Δ C t method was applied on qPCR data followed by division to gain a straightforward

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained
    Figure Legend Snippet: Methylation levels of LZTS1 locus; identical qPCR assays (fragments A-K) as for 5hmC assessment were used. Δ C t methodology was applied to minimize the experimental bias and varying DNA concentrations. ΔΔ C t values were obtained

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: To this end, 500 ng of DNA input of each sample was glucosylated with 5hmC glucosyltransferase to glu-5hmC for 6 hours at 37°C and digested with the GSRE Msp I, according to the manual of the Quest 5-hmC Detection Kit (Zymo Research). .. The amplification products of the multiplex PCR with 360 target DNA fragments from each sample were pooled in a 0.2-ml PCR tube and hybridized onto the CpG-360 microarray for analysis of 5hmC in 323 gene loci and 5mC in all gene loci.

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Converted DNA was purified and amplified using hot-start ZymoTaq and bisulfite-specific primer pairs ( ). .. Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research).

    Positive Control:

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research). .. Genomic DNA was divided into three groups: 1) Glycosylated and digested with a glucosyl-5hmC sensitive endonuclease, MspI [+GT]; 2) Unglucosylated and digested with MspI [-GT] (negative control); 3) unprocessed genomic DNA [untreated] (positive control).

    Quantitative RT-PCR:

    Article Title: Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis
    Article Snippet: DNA glucosylation and digestion was performed using the Quest 5-hmC Detection Kit (Zymo Research, Irvine, California, USA). .. Glucosylated DNA was digested with MspI at 37°C for 4 h. Global LINE-1 and local LINE-1 specific 5-hmc detection and quantification were performed by qRT-PCR, as described in .

    Real-time Polymerase Chain Reaction:

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system
    Article Snippet: Quantification of the 5hmC content of Ecat1 The 5hmC content of the Ecat1 gene was measured using the Quest 5-hmC Detection Kit (Zymo Research, Irvine). .. After glucosylation of 5hmC, the DNA was digested with a glucosyl-5hmC-sensitive restriction endonuclease (MspI) and then quantitative PCR was performed using Ecat1 -specific primers: 5′-GGAGAGCACATCCCACATCT-3′ and 5′-GTGAGCCAGATCAGTGAGCA-3′.

    Article Title: 5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes
    Article Snippet: Glycosylation of 5hmC Site-specific detection of 5hmC by glycosylation was done using the Quest 5-hmC detection kit (Zymo Research) following the manufacturer's instructions with modifications. .. The fraction of glycosylated and therefore protected Msp I sites as well as the fraction of 5mC- and 5hmC-sensitive sites (determined using Hpa II restriction) at specific gene loci were quantified by qPCR using primers described in [ ] or primers listed in Table S1 in Additional File .

    Article Title: TET2 is a Master Regulator of Smooth Muscle Cell Plasticity
    Article Snippet: Paragraph title: Glucosylation-coupled methylation-sensitivity qPCR (GlucMS-qPCR) ... Sequence specific detection of 5-hmC and 5-mC as a percentage of total C was performed using the Quest 5-hmC Detection Kit (Zymo Research) based on published methods.

    Article Title: The epigenetic control of E-box and Myc-dependent chromatin modifications regulate the licensing of lamin B2 origin during cell cycle
    Article Snippet: .. Detection and quantitation of 5-hmC by qPCR The Quest 5-hmC Detection Kit™-Lite (Zymo Research) was used for a sequence-specific detection of 5-hydroxymethylcytosine (5-hmC) as per supplier’s instructions. ..

    Article Title: Hypermethylation of the Micro-RNA 145 Promoter Is the Key Regulator for NLRP3 Inflammasome-Induced Activation and Plaque Formation
    Article Snippet: DNMT1 activity assay and 5-hydroxymethylcytosine detection DNMT1 activity and 5-hydroxymethylcytosine (5-hmC) level were detected by an EpiQuik DNA methyltransferase 1 activity assay kit (EpiGentek, Farmingdale, New York) and a Quest 5-hmC detection kit (Zymo Research, Irvine, California) to observe DNMT1 or TET2 activity, respectively. .. The results were detected by a microplate reader at 450 nm or by real-time polymerase chain reaction.

    Microarray:

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: Paragraph title: Identification of LZTS1 on Targeted Microarray ... To this end, 500 ng of DNA input of each sample was glucosylated with 5hmC glucosyltransferase to glu-5hmC for 6 hours at 37°C and digested with the GSRE Msp I, according to the manual of the Quest 5-hmC Detection Kit (Zymo Research).

    Quantitation Assay:

    Article Title: The epigenetic control of E-box and Myc-dependent chromatin modifications regulate the licensing of lamin B2 origin during cell cycle
    Article Snippet: .. Detection and quantitation of 5-hmC by qPCR The Quest 5-hmC Detection Kit™-Lite (Zymo Research) was used for a sequence-specific detection of 5-hydroxymethylcytosine (5-hmC) as per supplier’s instructions. ..

    Activity Assay:

    Article Title: Hypermethylation of the Micro-RNA 145 Promoter Is the Key Regulator for NLRP3 Inflammasome-Induced Activation and Plaque Formation
    Article Snippet: .. DNMT1 activity assay and 5-hydroxymethylcytosine detection DNMT1 activity and 5-hydroxymethylcytosine (5-hmC) level were detected by an EpiQuik DNA methyltransferase 1 activity assay kit (EpiGentek, Farmingdale, New York) and a Quest 5-hmC detection kit (Zymo Research, Irvine, California) to observe DNMT1 or TET2 activity, respectively. .. The results were detected by a microplate reader at 450 nm or by real-time polymerase chain reaction.

    Expressing:

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: We fully agree with the reviewer that this is indeed an interesting point, and we currently do not have an explanation for why only a subset of H3K27me3-marked CGIs would be targeted by ectopic Dnmt3b expression. .. To explore this, we randomly selected 6 CpG sites that display low or high methylation levels in bulk RRBS from the liver of adult mice after dox induction for 3 months and checked the hydroxymethylation level of them using the Quest 5-hmC Detection Kit (Zymo Research).

    Genome Wide:

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: .. For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions. .. Briefly, the genomic DNA was treated with 5hmC glucosyltransferase enzyme, which specifically tags the 5hmC in DNA with a glucose moiety yielding a modified base, glucosyl-5hmC.

    Modification:

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system
    Article Snippet: Quantification of the 5hmC content of Ecat1 The 5hmC content of the Ecat1 gene was measured using the Quest 5-hmC Detection Kit (Zymo Research, Irvine). .. This kit enables sequence-specific detection of 5hmC within DNA; utilizing a 5hmC glucosyltransferase, 5hmC in DNA is specifically tagged with a glucose moiety yielding the modified base glucosyl-5hmC.

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions. .. Briefly, the genomic DNA was treated with 5hmC glucosyltransferase enzyme, which specifically tags the 5hmC in DNA with a glucose moiety yielding a modified base, glucosyl-5hmC.

    Over Expression:

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: To explore this, we randomly selected 6 CpG sites that display low or high methylation levels in bulk RRBS from the liver of adult mice after dox induction for 3 months and checked the hydroxymethylation level of them using the Quest 5-hmC Detection Kit (Zymo Research). .. No significant increase in hydroxymethylation level was observed for these CpG sites in the liver with Dnmt3b overexpression compared to the control mice.

    Transfection:

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: Cells were lysed 96 h after transfection in Trizol reagent, and both total RNA and genomic DNA were prepared according to manufacturer's instructions. .. For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions.

    other:

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: Five hundred nanograms of genomic DNA from patient samples, standardized controls, and blood was applied to the Quest 5hmC Detection Kit.

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: To assess hydroxymethylation at selected CpGs, we used the Quest 5-hmC Detection Kit (Zymo Research) according to manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: To this end, 500 ng of DNA input of each sample was glucosylated with 5hmC glucosyltransferase to glu-5hmC for 6 hours at 37°C and digested with the GSRE Msp I, according to the manual of the Quest 5-hmC Detection Kit (Zymo Research). .. The amplification products of the multiplex PCR with 360 target DNA fragments from each sample were pooled in a 0.2-ml PCR tube and hybridized onto the CpG-360 microarray for analysis of 5hmC in 323 gene loci and 5mC in all gene loci.

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: PCR amplicons (~300bp) were either directly sequenced or sub-cloned for sequencing. .. Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research).

    Methylation:

    Article Title: TET2 is a Master Regulator of Smooth Muscle Cell Plasticity
    Article Snippet: Paragraph title: Glucosylation-coupled methylation-sensitivity qPCR (GlucMS-qPCR) ... Sequence specific detection of 5-hmC and 5-mC as a percentage of total C was performed using the Quest 5-hmC Detection Kit (Zymo Research) based on published methods.

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: Glucosylation of 5hmC was performed using the Quest 5hmC Detection Kit (Zymo Research, Irvine, CA), according to manufacturer's instructions. .. To distinguish between 5-hydroxymethylated DNA and methylated or unmethylated DNA, each reaction was split into two reactions containing the 5hmC glucosyltransferase enzyme or no enzyme, respectively.

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: .. To explore this, we randomly selected 6 CpG sites that display low or high methylation levels in bulk RRBS from the liver of adult mice after dox induction for 3 months and checked the hydroxymethylation level of them using the Quest 5-hmC Detection Kit (Zymo Research). .. No significant increase in hydroxymethylation level was observed for these CpG sites in the liver with Dnmt3b overexpression compared to the control mice.

    Article Title: DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria
    Article Snippet: The presence of 5hmC at Gla1 restriction sites was determined using a Quest 5hmC Detection Kit (Zymo Research) as described by the manufacturer using 80 ng mtDNA or total cellular DNA. .. Gla1 cleaves DNA only when restriction-site cytosines are methylated or hydroxymethylated; glucosylation of 5hmC residues results in protection from Gla1 cleavage.

    Multiplex Assay:

    Article Title: Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer 1 Gene Is Reduced in Breast Cancer 1 2
    Article Snippet: To this end, 500 ng of DNA input of each sample was glucosylated with 5hmC glucosyltransferase to glu-5hmC for 6 hours at 37°C and digested with the GSRE Msp I, according to the manual of the Quest 5-hmC Detection Kit (Zymo Research). .. The amplification products of the multiplex PCR with 360 target DNA fragments from each sample were pooled in a 0.2-ml PCR tube and hybridized onto the CpG-360 microarray for analysis of 5hmC in 323 gene loci and 5mC in all gene loci.

    Mouse Assay:

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: .. To explore this, we randomly selected 6 CpG sites that display low or high methylation levels in bulk RRBS from the liver of adult mice after dox induction for 3 months and checked the hydroxymethylation level of them using the Quest 5-hmC Detection Kit (Zymo Research). .. No significant increase in hydroxymethylation level was observed for these CpG sites in the liver with Dnmt3b overexpression compared to the control mice.

    Dot Blot:

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: Paragraph title: 5hmC slot blot, and genome-wide mapping and analysis of 5hmC ... For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions.

    Sequencing:

    Article Title: Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system
    Article Snippet: Quantification of the 5hmC content of Ecat1 The 5hmC content of the Ecat1 gene was measured using the Quest 5-hmC Detection Kit (Zymo Research, Irvine). .. This kit enables sequence-specific detection of 5hmC within DNA; utilizing a 5hmC glucosyltransferase, 5hmC in DNA is specifically tagged with a glucose moiety yielding the modified base glucosyl-5hmC.

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions. .. MspI efficiently cleaves the DNA when cytosine, 5mC, or 5hmC but not glucysyl-5hmC is located at the inner C position within its recognition sequence (i.e. CC GG).

    Article Title: TET2 is a Master Regulator of Smooth Muscle Cell Plasticity
    Article Snippet: .. Sequence specific detection of 5-hmC and 5-mC as a percentage of total C was performed using the Quest 5-hmC Detection Kit (Zymo Research) based on published methods. ..

    Article Title: DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria
    Article Snippet: Paragraph title: Sequence-Specific Detection of 5hmC. ... The presence of 5hmC at Gla1 restriction sites was determined using a Quest 5hmC Detection Kit (Zymo Research) as described by the manufacturer using 80 ng mtDNA or total cellular DNA.

    Article Title: The epigenetic control of E-box and Myc-dependent chromatin modifications regulate the licensing of lamin B2 origin during cell cycle
    Article Snippet: .. Detection and quantitation of 5-hmC by qPCR The Quest 5-hmC Detection Kit™-Lite (Zymo Research) was used for a sequence-specific detection of 5-hydroxymethylcytosine (5-hmC) as per supplier’s instructions. ..

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Sequencing traces were analyzed using QUMA (RIKEN, Japan). .. Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research).

    Purification:

    Article Title: Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination
    Article Snippet: 5hmC quantification DNA was purified from each HeLa cell line using organic extraction, followed by an ethanol precipitation. .. The amount of 5hmC present in each DNA sample was quantified using a Quest 5hmC detection kit (Zymo Research) according to the manufacturer's instructions.

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Converted DNA was purified and amplified using hot-start ZymoTaq and bisulfite-specific primer pairs ( ). .. Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research).

    Software:

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity
    Article Snippet: Relative signal strengths of 5hmC bands were quantified using the Image-J software ( http://imagej.nih.gov/ij/ ). .. For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions.

    Negative Control:

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research). .. Genomic DNA was divided into three groups: 1) Glycosylated and digested with a glucosyl-5hmC sensitive endonuclease, MspI [+GT]; 2) Unglucosylated and digested with MspI [-GT] (negative control); 3) unprocessed genomic DNA [untreated] (positive control).

    Ethanol Precipitation:

    Article Title: Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination
    Article Snippet: 5hmC quantification DNA was purified from each HeLa cell line using organic extraction, followed by an ethanol precipitation. .. The amount of 5hmC present in each DNA sample was quantified using a Quest 5hmC detection kit (Zymo Research) according to the manufacturer's instructions.

    DNA Methylation Assay:

    Article Title: Targets and genomic constraints of ectopic Dnmt3b expression
    Article Snippet: 4) It seems important to verify, at least for a few loci, that Dnmt3b-induced gain of DNA methylation quantified by RRBS corresponds to methylated but not hydroxymethylated cytosines. .. To explore this, we randomly selected 6 CpG sites that display low or high methylation levels in bulk RRBS from the liver of adult mice after dox induction for 3 months and checked the hydroxymethylation level of them using the Quest 5-hmC Detection Kit (Zymo Research).

    DNA Purification:

    Article Title: Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways
    Article Snippet: Locus-specific 5hmC quantification was performed using the Quest 5hmC Detection kit (Zymo Research). .. Briefly, genomic DNA was extracted at 72hpf using Purelink Genomic DNA purification kit (Invitrogen).

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    Zymo Research quest 5 hmc detection kit
    Quest 5 Hmc Detection Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quest 5 hmc detection kit/product/Zymo Research
    Average 99 stars, based on 10 article reviews
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    quest 5 hmc detection kit - by Bioz Stars, 2020-04
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    93
    Zymo Research quest 5 hmc detection kit lite
    Quest 5 Hmc Detection Kit Lite, supplied by Zymo Research, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quest 5 hmc detection kit lite/product/Zymo Research
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    quest 5 hmc detection kit lite - by Bioz Stars, 2020-04
    93/100 stars
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