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  • u251  (ATCC)
    86
    ATCC u251
    KAT2A coupled with the α-KGDH complex regulates H3K79 succinylation and gene expression a , H3K79 is succinylated. Purified wild-type or mutated histone H3 was incubated with purified KAT2A and succinyl-CoA. Mass spectrometric analysis of a tryptic fragment of histone H3 at monoisotopic m / z 718.35706 Da (+ 0.21 milli-mass unit (m.m.u.)/+ 0.29 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 51, and the expectation value ( P ) was 5.5 × 10 −3 . b , Analyses of the specificity of the antibody against H3K79 succinylation. Synthetic histone H3 peptides with or without H3K79 succinylation were used to test the specificity of the antibody against H3K79 succinylation. Representative images of triplicate experiments are shown. MW, molecular weight. c , Identification of histone H3 K79 succinylation in <t>U251</t> cells. Histones were extracted from U251 cells. Mass spectrometric analysis of a tryptic fragment of histone H3 extracts from U251 cells at monoisotopic m / z 718.35559 Da (− 1.26 m.m.u./ − 1.75 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 40, and the expectation value ( P ) was 2.5 × 10 −2 . d , Semi-quantitative comparison of posttranslational modifications of histone H3 with or without KAT2A depletion. Histones were extracted from U251 cells with or without KAT2A depletion. Mass spectrometric analyses identified the specific peptides of H3K14 acetylation (KSTGGKacAPR), H3K24 acetylation (KQLATKacAAR), H3K79 succinylation (EIAQDFKsuccTDLR), and H3K122 acetylation (RVTIMPKacDIQLAR). The succinylated H3K79 peptide was identified in the histone extracts from U251 cells in the absence but not presence of KAT2A depletion (Mascot IonScore, 40; expectation value, 2.5 × 10 −2 ). The peptides of H3K14ac, H3K24ac, and H3K122ac were identified in histone extracts from U251 cells with or without KAT2A depletion (Mascot IonScore > 30; expectation value
    U251, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore u251 cells
    IsoA (4 mg/ml) treatment decreases TGFβ and regulates EMT markers in glioma cells. ( A, B ) IsoA treatment decreased TGFβ mRNA ( A ) and protein ( B ) expression levels in <t>U251</t> cells. ( C, D ) IsoA treatment decreases Fibronectin, Vimentin, and increased E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells.
    U251 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    ATCC u251 cells
    MiR-23a promotes invasion of GBM cells. a The Matrigel invasion assays were performed using six human GBM cell lines: LN443, U373, LN308, U87, <t>U251,</t> and KMG4. * P
    U251 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Size 1 vial Price 672 0 Molecule Name u 251 mg glioblastoma cell line
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    U 373 MG is identical to U 251 MG U 251 MG cell line was derived from a malignant glioblastoma tumour by explant technique
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    U 251 Whole Cell Lysate W09 001 GY4
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    Category Cell Products Cell Line U251 Cell Line Size 1×10⁶ cells vial Price 349
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    Image Search Results


    KAT2A coupled with the α-KGDH complex regulates H3K79 succinylation and gene expression a , H3K79 is succinylated. Purified wild-type or mutated histone H3 was incubated with purified KAT2A and succinyl-CoA. Mass spectrometric analysis of a tryptic fragment of histone H3 at monoisotopic m / z 718.35706 Da (+ 0.21 milli-mass unit (m.m.u.)/+ 0.29 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 51, and the expectation value ( P ) was 5.5 × 10 −3 . b , Analyses of the specificity of the antibody against H3K79 succinylation. Synthetic histone H3 peptides with or without H3K79 succinylation were used to test the specificity of the antibody against H3K79 succinylation. Representative images of triplicate experiments are shown. MW, molecular weight. c , Identification of histone H3 K79 succinylation in U251 cells. Histones were extracted from U251 cells. Mass spectrometric analysis of a tryptic fragment of histone H3 extracts from U251 cells at monoisotopic m / z 718.35559 Da (− 1.26 m.m.u./ − 1.75 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 40, and the expectation value ( P ) was 2.5 × 10 −2 . d , Semi-quantitative comparison of posttranslational modifications of histone H3 with or without KAT2A depletion. Histones were extracted from U251 cells with or without KAT2A depletion. Mass spectrometric analyses identified the specific peptides of H3K14 acetylation (KSTGGKacAPR), H3K24 acetylation (KQLATKacAAR), H3K79 succinylation (EIAQDFKsuccTDLR), and H3K122 acetylation (RVTIMPKacDIQLAR). The succinylated H3K79 peptide was identified in the histone extracts from U251 cells in the absence but not presence of KAT2A depletion (Mascot IonScore, 40; expectation value, 2.5 × 10 −2 ). The peptides of H3K14ac, H3K24ac, and H3K122ac were identified in histone extracts from U251 cells with or without KAT2A depletion (Mascot IonScore > 30; expectation value

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: KAT2A coupled with the α-KGDH complex regulates H3K79 succinylation and gene expression a , H3K79 is succinylated. Purified wild-type or mutated histone H3 was incubated with purified KAT2A and succinyl-CoA. Mass spectrometric analysis of a tryptic fragment of histone H3 at monoisotopic m / z 718.35706 Da (+ 0.21 milli-mass unit (m.m.u.)/+ 0.29 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 51, and the expectation value ( P ) was 5.5 × 10 −3 . b , Analyses of the specificity of the antibody against H3K79 succinylation. Synthetic histone H3 peptides with or without H3K79 succinylation were used to test the specificity of the antibody against H3K79 succinylation. Representative images of triplicate experiments are shown. MW, molecular weight. c , Identification of histone H3 K79 succinylation in U251 cells. Histones were extracted from U251 cells. Mass spectrometric analysis of a tryptic fragment of histone H3 extracts from U251 cells at monoisotopic m / z 718.35559 Da (− 1.26 m.m.u./ − 1.75 p.p.m.) matched with the doubly charged peptide 73-EIAQDFKTDLR-83 with K7-succinyl (100.01604 Da), suggesting that H3K79 was succinylated. The IonScore (Mascot) was 40, and the expectation value ( P ) was 2.5 × 10 −2 . d , Semi-quantitative comparison of posttranslational modifications of histone H3 with or without KAT2A depletion. Histones were extracted from U251 cells with or without KAT2A depletion. Mass spectrometric analyses identified the specific peptides of H3K14 acetylation (KSTGGKacAPR), H3K24 acetylation (KQLATKacAAR), H3K79 succinylation (EIAQDFKsuccTDLR), and H3K122 acetylation (RVTIMPKacDIQLAR). The succinylated H3K79 peptide was identified in the histone extracts from U251 cells in the absence but not presence of KAT2A depletion (Mascot IonScore, 40; expectation value, 2.5 × 10 −2 ). The peptides of H3K14ac, H3K24ac, and H3K122ac were identified in histone extracts from U251 cells with or without KAT2A depletion (Mascot IonScore > 30; expectation value

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Expressing, Purification, Incubation, Molecular Weight

    α-KGDH-coupled KAT2A regulates H3K79 succinylation and gene expression a – d , g , h , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , H3K79 is succinylated. Purified wild-type histone H3 or H3(K79R) was incubated with succinyl-CoA and purified wild-type or inactive KAT2A. b , KAT2A-depdendent H3K79 succinylation. KAT2A shRNA was expressed in U87 and U251 cells. c , α-KGDH provides succinyl-CoA for H3K79 succinylation. Purified KAT2A, histone H3, CoA, α-ketoglutarate, and NAD + were incubated with or without immunoprecipitated wild-type Flag–rOGDH or Flag–rOGDH(P459A/Y460A) from 293 cells. Mut, mutant. d , Nuclear localization of α-KGDH is involved in H3K79 succinylation. U251 cells with depleted endogenous DLST and reconstituted wild-type V5–rDLST or V5–rDLST NLS mutant were analysed by immunoblotting assay. e , Average genome-wide occupancies of H3K79 succinylation (red), KAT2A (blue), and α-KGDH (OGDH, black) around the transcription start site (TSS). f , KAT2A promotes H3K79 succinylation at the indicated gene promoters. ChIP assay with an anti-H3K79 succinylation antibody and quantitative PCR with primers against the promoter regions of PIK3R1 , JUN , and PRKDC in U251 cells with or without KAT2A depletion were performed. Two-sided t -test analyses were conducted. The data are presented as the means ± s.d. of three independent experiments ( n = 3). * P

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: α-KGDH-coupled KAT2A regulates H3K79 succinylation and gene expression a – d , g , h , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , H3K79 is succinylated. Purified wild-type histone H3 or H3(K79R) was incubated with succinyl-CoA and purified wild-type or inactive KAT2A. b , KAT2A-depdendent H3K79 succinylation. KAT2A shRNA was expressed in U87 and U251 cells. c , α-KGDH provides succinyl-CoA for H3K79 succinylation. Purified KAT2A, histone H3, CoA, α-ketoglutarate, and NAD + were incubated with or without immunoprecipitated wild-type Flag–rOGDH or Flag–rOGDH(P459A/Y460A) from 293 cells. Mut, mutant. d , Nuclear localization of α-KGDH is involved in H3K79 succinylation. U251 cells with depleted endogenous DLST and reconstituted wild-type V5–rDLST or V5–rDLST NLS mutant were analysed by immunoblotting assay. e , Average genome-wide occupancies of H3K79 succinylation (red), KAT2A (blue), and α-KGDH (OGDH, black) around the transcription start site (TSS). f , KAT2A promotes H3K79 succinylation at the indicated gene promoters. ChIP assay with an anti-H3K79 succinylation antibody and quantitative PCR with primers against the promoter regions of PIK3R1 , JUN , and PRKDC in U251 cells with or without KAT2A depletion were performed. Two-sided t -test analyses were conducted. The data are presented as the means ± s.d. of three independent experiments ( n = 3). * P

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Expressing, Purification, Incubation, shRNA, Immunoprecipitation, Mutagenesis, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    KAT2A associates with α-KGDH and acts as a histone succinyltransferase a , rDLST NLS mutation does not affect the enzymatic activity of α-KGDH. Wild-type His–rDLST or His–rDLST NLS mutant expressed in 293 cells was precipitated using Ni-NTA agarose beads. The enzymatic activity of α-KGDH was measured. Two-sided t -tests were conducted. The data are presented as the means ± s.d. from five independent experiments ( n = 5). b , rDLST NLS mutant expression does not affect cellular levels of acetyl-CoA or succinyl-CoA. The levels of acetyl-CoA and succinyl-CoA in U251 cells with depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were measured by LC–MS/MS. Two-sided t -tests were conducted. The data are presented as the means ± s.d. from three independent experiments ( n = 3). c , rDLST NLS mutant expression does not affect total cellular protein succinylation. Lysine succinylation of whole-cell lysate of U251 cells expressing wild-type V5–rDLST or V5–rDLST NLS mutant was analysed by immunoblotting analysis with an anti-succinyl-lysine antibody. The protein levels of each sample were shown by Ponceau S staining. Representative images of triplicate experiments are shown. d , Quantification of the colocalization of KAT2A, α-KGDH complex, and histone H3. Immunofluorescent analyses were performed with anti-DLST (red), anti-KAT2A (blue), and anti-histone H3 (green) antibodies. The Pearson’s R value is used to present the extent of co-localization of two different targets. The random co-localization tests were conducted by 90° clockwise rotation of KAT2A (blue channel) and 180° clockwise rotation of histone H3 (green channel). Two-sided t -tests were conducted. Statistical analysis for 79 individual cells ( n = 79) was performed. * P

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: KAT2A associates with α-KGDH and acts as a histone succinyltransferase a , rDLST NLS mutation does not affect the enzymatic activity of α-KGDH. Wild-type His–rDLST or His–rDLST NLS mutant expressed in 293 cells was precipitated using Ni-NTA agarose beads. The enzymatic activity of α-KGDH was measured. Two-sided t -tests were conducted. The data are presented as the means ± s.d. from five independent experiments ( n = 5). b , rDLST NLS mutant expression does not affect cellular levels of acetyl-CoA or succinyl-CoA. The levels of acetyl-CoA and succinyl-CoA in U251 cells with depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were measured by LC–MS/MS. Two-sided t -tests were conducted. The data are presented as the means ± s.d. from three independent experiments ( n = 3). c , rDLST NLS mutant expression does not affect total cellular protein succinylation. Lysine succinylation of whole-cell lysate of U251 cells expressing wild-type V5–rDLST or V5–rDLST NLS mutant was analysed by immunoblotting analysis with an anti-succinyl-lysine antibody. The protein levels of each sample were shown by Ponceau S staining. Representative images of triplicate experiments are shown. d , Quantification of the colocalization of KAT2A, α-KGDH complex, and histone H3. Immunofluorescent analyses were performed with anti-DLST (red), anti-KAT2A (blue), and anti-histone H3 (green) antibodies. The Pearson’s R value is used to present the extent of co-localization of two different targets. The random co-localization tests were conducted by 90° clockwise rotation of KAT2A (blue channel) and 180° clockwise rotation of histone H3 (green channel). Two-sided t -tests were conducted. Statistical analysis for 79 individual cells ( n = 79) was performed. * P

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Mutagenesis, Activity Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining

    KAT2A interacts with the α-KGDH complex in the nucleus a , OGDH binds to KAT2A. KAT2A was immunoprecipitated from U251 cells using an anti-KAT2A antibody. The immunoprecipitate was analysed by mass spectrometry. A specific peptide (920-DMVGQVAIT-929) of OGDH was identified. The IonScore is 30, the q value is 0.004, and the Exp value ( P ) is 0.1792. One-sided t -tests were conducted. Representative results of two experiments are shown. b , Quantitative analyses of α-KGDH subunits in the nucleus. Immunoblotting analyses of α-KGDH subunits in the nucleus extraction of U251 cells were performed with the indicated antibodies displayed in . The quantitative standard curves were used to determine the percentage of nuclear OGDH, DLST, and DLD. The x -axis presents the intensities of quantified proteins detected by immunoblotting analysis, and the y -axis presents the percentage of whole-cell lysate corresponding to each quantified protein. The percentages of nuclear OGDH, DLST, and DLD were calculated using the equations displayed above the standard curves. Representative images of triplicate experiments are shown. The R 2 values represent the relationship between log Y and X to the linear model (log Y = log[ A + B ] × X ). Y , percentage of whole cell lysate; X , intensity. c , Nuclear distribution of the α-KGDH complex. Immunofluorescent staining of U251 cells was performed with anti-OGDH, anti-DLST, or anti-DLD antibodies (green). The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. Representative images of more than 50 cells are shown. The majority of the α-KGDH complex overlapped with the mitochondrial protein Tom20, indicating primary mitochondrial localization of the α-KGDH complex. d , Quantitative analyses of the interaction between KAT2A and α-KGDH subunits. Immunoprecipitation analyses of U251 cell nuclei with anti-KAT2A or anti-OGDH antibodies were performed. KAT2A-or OGDH-associated proteins were analysed via immunoblotting analyses. Representative images of triplicate experiments are shown. e , f , KAT2A is not localized in mitochondria. e , Immunoblotting analyses of the mitochondria extraction and total cell lysate of U251 cells were performed with the indicated antibodies. Representative images of triplicate experiments are shown. f , Immunofluorescent analyses of U251 cells were performed with DAPI (blue) and anti-KAT2A (green) and anti- TOM20 (red) antibodies. Representative images of more than 50 cells are shown. g , OGDH depletion, which does not affect the interaction between DLST and DLD, inhibits the binding of DLST to KAT2A. OGDH was depleted in U251 cells. Immunoprecipitation analyses with an anti-DLST antibody were followed by immunoblotting analyses with the indicated antibodies. Representative images of triplicate experiments are shown. h , i , The NLS of DLST is required for nuclear distribution of the α-KGDH complex. DLST was depleted from U87 cells, which were reconstituted with expression of wild-type rDLST or the rDLST(R224A/K226E) NLS mutant. h , Immunofluorescent staining was performed with an anti- DLST or anti-OGDH antibody. The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. i , Immunoblotting analyses were performed with the indicated antibodies. Mitochondrial COX IV and IDH2 and nuclear PCNA were used to show lack of mitochondrial contamination of nuclear fractions and loading controls. Representative images of triplicate experiments are shown. Fig. 1b

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: KAT2A interacts with the α-KGDH complex in the nucleus a , OGDH binds to KAT2A. KAT2A was immunoprecipitated from U251 cells using an anti-KAT2A antibody. The immunoprecipitate was analysed by mass spectrometry. A specific peptide (920-DMVGQVAIT-929) of OGDH was identified. The IonScore is 30, the q value is 0.004, and the Exp value ( P ) is 0.1792. One-sided t -tests were conducted. Representative results of two experiments are shown. b , Quantitative analyses of α-KGDH subunits in the nucleus. Immunoblotting analyses of α-KGDH subunits in the nucleus extraction of U251 cells were performed with the indicated antibodies displayed in . The quantitative standard curves were used to determine the percentage of nuclear OGDH, DLST, and DLD. The x -axis presents the intensities of quantified proteins detected by immunoblotting analysis, and the y -axis presents the percentage of whole-cell lysate corresponding to each quantified protein. The percentages of nuclear OGDH, DLST, and DLD were calculated using the equations displayed above the standard curves. Representative images of triplicate experiments are shown. The R 2 values represent the relationship between log Y and X to the linear model (log Y = log[ A + B ] × X ). Y , percentage of whole cell lysate; X , intensity. c , Nuclear distribution of the α-KGDH complex. Immunofluorescent staining of U251 cells was performed with anti-OGDH, anti-DLST, or anti-DLD antibodies (green). The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. Representative images of more than 50 cells are shown. The majority of the α-KGDH complex overlapped with the mitochondrial protein Tom20, indicating primary mitochondrial localization of the α-KGDH complex. d , Quantitative analyses of the interaction between KAT2A and α-KGDH subunits. Immunoprecipitation analyses of U251 cell nuclei with anti-KAT2A or anti-OGDH antibodies were performed. KAT2A-or OGDH-associated proteins were analysed via immunoblotting analyses. Representative images of triplicate experiments are shown. e , f , KAT2A is not localized in mitochondria. e , Immunoblotting analyses of the mitochondria extraction and total cell lysate of U251 cells were performed with the indicated antibodies. Representative images of triplicate experiments are shown. f , Immunofluorescent analyses of U251 cells were performed with DAPI (blue) and anti-KAT2A (green) and anti- TOM20 (red) antibodies. Representative images of more than 50 cells are shown. g , OGDH depletion, which does not affect the interaction between DLST and DLD, inhibits the binding of DLST to KAT2A. OGDH was depleted in U251 cells. Immunoprecipitation analyses with an anti-DLST antibody were followed by immunoblotting analyses with the indicated antibodies. Representative images of triplicate experiments are shown. h , i , The NLS of DLST is required for nuclear distribution of the α-KGDH complex. DLST was depleted from U87 cells, which were reconstituted with expression of wild-type rDLST or the rDLST(R224A/K226E) NLS mutant. h , Immunofluorescent staining was performed with an anti- DLST or anti-OGDH antibody. The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. i , Immunoblotting analyses were performed with the indicated antibodies. Mitochondrial COX IV and IDH2 and nuclear PCNA were used to show lack of mitochondrial contamination of nuclear fractions and loading controls. Representative images of triplicate experiments are shown. Fig. 1b

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Immunoprecipitation, Mass Spectrometry, Staining, Binding Assay, Expressing, Mutagenesis

    Selected cell signalling pathways enriched with the genes with H3K79-succinylated promoter regions a , H3K79 succinylation regulates critical cellular signalling pathways. Hypergeometric tests selected H3K79 succinylation-enriched signalling pathways in U251 cells with DLST(R224A/K226E) or KAT2A(Y645A) mutant expression. y -axis: level of significance from RNA-seq data, which takes the form of (− log 10 P ) × reg, where reg = − 1, indicating that the pathway is enriched with downregulated genes based on the comparison of mutant with wild-type proteins. One-sided hypergeometric test for overrepresentation was conducted. The data are from one RNA-seq sample. b , c , Combined view of ChIP–seq and RNA-seq data revealing histone H3K79 succinylation-enriched pathways that were significantly suppressed by the expression of V5–rDLST(R224A/K226E) ( b ) or Flag–rKAT2A(Y645A) ( c ). Significantly downregulated pathways were identified via hypergeometric tests (blue nodes, P

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: Selected cell signalling pathways enriched with the genes with H3K79-succinylated promoter regions a , H3K79 succinylation regulates critical cellular signalling pathways. Hypergeometric tests selected H3K79 succinylation-enriched signalling pathways in U251 cells with DLST(R224A/K226E) or KAT2A(Y645A) mutant expression. y -axis: level of significance from RNA-seq data, which takes the form of (− log 10 P ) × reg, where reg = − 1, indicating that the pathway is enriched with downregulated genes based on the comparison of mutant with wild-type proteins. One-sided hypergeometric test for overrepresentation was conducted. The data are from one RNA-seq sample. b , c , Combined view of ChIP–seq and RNA-seq data revealing histone H3K79 succinylation-enriched pathways that were significantly suppressed by the expression of V5–rDLST(R224A/K226E) ( b ) or Flag–rKAT2A(Y645A) ( c ). Significantly downregulated pathways were identified via hypergeometric tests (blue nodes, P

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Mutagenesis, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation

    KAT2A acts as a histone succinyltransferase a – c , f , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , Nuclear DLST promotes histone H3 succinylation. Histones were extracted from U251 cells with depleted endogenous DLST and reconstituted expression of wild-type rDLST or the rDLST NLS mutant. The total histone acetylation and succinylation levels were analysed. The histone protein levels were analysed with GelCode Blue staining. b , KAT2A regulates histone H3 succinylation. Histone H3 proteins were immunoprecipitated from U251 cells with or without KAT2A depletion (by shRNA). c , KAT2A succinylates histone H3. KAT2A-mediated histone H3 succinylation was analysed by mixing purified KAT2A, histone H3, and succinyl-CoA. Heat-inactived KAT2A was used as a negative control. d , Superimposition of the KAT2A apo, KAT2A–succidnyl-CoA complex, and KAT2A–acetyl-CoA complex structures. Models show KAT2A in ribbons, succinyl-CoA in red sticks, and acetyl-CoA in blue sticks. The three loop regions are highlighted, with the apo structure in green, KAT2A–acetyl-CoA complex in blue, and KAT2A–succinyl-CoA complex in red. The rest of the structures are shown in white, grey, and black for apo, the KAT2A-acetyl–CoA complex, and the KAT2A-succinyl–CoA complex, respectively. The three loops, secondary structure elements, and the N and C termini of KAT2A are labelled. The two extra carbons in the succinyl group compared to the acetyl group are highlighted with red dashes. e , Interactions between succinyl-CoA and loop 3 of KAT2A. KAT2A is rainbow-coloured, with the N terminus in blue and the C terminus in red. Tyr645, Ala648, and the succinyl-CoA molecule are shown as sticks. f , KAT2A Y645 is critical for histone H3 succinylation. U251 cells with depleted endogenous KAT2A and reconstituted expression of wild-type Flag–rKAT2A or Flag–rKAT2A(Y645A) were analysed by immunoblotting assay.

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: KAT2A acts as a histone succinyltransferase a – c , f , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , Nuclear DLST promotes histone H3 succinylation. Histones were extracted from U251 cells with depleted endogenous DLST and reconstituted expression of wild-type rDLST or the rDLST NLS mutant. The total histone acetylation and succinylation levels were analysed. The histone protein levels were analysed with GelCode Blue staining. b , KAT2A regulates histone H3 succinylation. Histone H3 proteins were immunoprecipitated from U251 cells with or without KAT2A depletion (by shRNA). c , KAT2A succinylates histone H3. KAT2A-mediated histone H3 succinylation was analysed by mixing purified KAT2A, histone H3, and succinyl-CoA. Heat-inactived KAT2A was used as a negative control. d , Superimposition of the KAT2A apo, KAT2A–succidnyl-CoA complex, and KAT2A–acetyl-CoA complex structures. Models show KAT2A in ribbons, succinyl-CoA in red sticks, and acetyl-CoA in blue sticks. The three loop regions are highlighted, with the apo structure in green, KAT2A–acetyl-CoA complex in blue, and KAT2A–succinyl-CoA complex in red. The rest of the structures are shown in white, grey, and black for apo, the KAT2A-acetyl–CoA complex, and the KAT2A-succinyl–CoA complex, respectively. The three loops, secondary structure elements, and the N and C termini of KAT2A are labelled. The two extra carbons in the succinyl group compared to the acetyl group are highlighted with red dashes. e , Interactions between succinyl-CoA and loop 3 of KAT2A. KAT2A is rainbow-coloured, with the N terminus in blue and the C terminus in red. Tyr645, Ala648, and the succinyl-CoA molecule are shown as sticks. f , KAT2A Y645 is critical for histone H3 succinylation. U251 cells with depleted endogenous KAT2A and reconstituted expression of wild-type Flag–rKAT2A or Flag–rKAT2A(Y645A) were analysed by immunoblotting assay.

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Expressing, Mutagenesis, Staining, Immunoprecipitation, shRNA, Purification, Negative Control

    α-KGDH-coupled KAT2A promotes tumour growth a , α-KGDH-coupled KAT2A promotes tumour cell proliferation. A total of 10 4 U251 cells with depleted endogenous KAT2A and reconstituted expression of wild-type Flag–rKAT2A or Flag–rKAT2A(Y645A) or depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were plated. The cells were collected and counted daily for nine days. The data are presented as the means ± s.d. from three independent experiments ( n = 3). b , α-KGDH-coupled KAT2A promotes tumour growth. U87 cells with depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were intracranially injected into athymic nude mice. Representative haematoxylin and eosin-stained coronal brain sections are shown. Tumour volumes were calculated. Two-sided t -tests were conducted. Data represent the means ± s.d. of 5 mice ( n = 5). * P

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: α-KGDH-coupled KAT2A promotes tumour growth a , α-KGDH-coupled KAT2A promotes tumour cell proliferation. A total of 10 4 U251 cells with depleted endogenous KAT2A and reconstituted expression of wild-type Flag–rKAT2A or Flag–rKAT2A(Y645A) or depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were plated. The cells were collected and counted daily for nine days. The data are presented as the means ± s.d. from three independent experiments ( n = 3). b , α-KGDH-coupled KAT2A promotes tumour growth. U87 cells with depleted endogenous DLST and reconstituted expression of wild-type V5–rDLST or V5–rDLST NLS mutant were intracranially injected into athymic nude mice. Representative haematoxylin and eosin-stained coronal brain sections are shown. Tumour volumes were calculated. Two-sided t -tests were conducted. Data represent the means ± s.d. of 5 mice ( n = 5). * P

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Expressing, Mutagenesis, Injection, Mouse Assay, Staining

    KAT2A interacts with the α-KGDH complex in the nucleus a – d , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , KAT2A binds to the α-KGDH complex. Immunoprecipitation analyses of U251 cells with an anti-KAT2A antibody were performed. KAT2A-associated proteins were analysed by immunoblotting. b , Nuclear localization of α-KGDH. Immunoblotting analyses of α-KGDH subunits in extracted nuclei of U251 cells were performed. c , OGDH binds to KAT2A. The α-KGDH complex in U251 cells with or without depletion of OGDH, DLST, or DLD using short hairpin RNA (shOGDH, shDLST or shDLD, respectively) was immunoprecipitated with the indicated antibodies and incubated with bacterially purified full-length His–KAT2A protein. d , DLST is required for nuclear distribution of the α-KGDH complex. DLST was depleted from U251 cells, which were reconstituted with expression of RNA interference-resistant (r) wild-type (WT) V5–rDLST or V5–rDLST(R224A/K226E) mutant (V5–rDLST NLS mut). Immunoblotting analyses were performed. Mitochondrial COX IV and IDH2 and nuclear proliferating cell nuclear antigen (PCNA) were used as controls.

    Journal: Nature

    Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase

    doi: 10.1038/nature25003

    Figure Lengend Snippet: KAT2A interacts with the α-KGDH complex in the nucleus a – d , Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown. a , KAT2A binds to the α-KGDH complex. Immunoprecipitation analyses of U251 cells with an anti-KAT2A antibody were performed. KAT2A-associated proteins were analysed by immunoblotting. b , Nuclear localization of α-KGDH. Immunoblotting analyses of α-KGDH subunits in extracted nuclei of U251 cells were performed. c , OGDH binds to KAT2A. The α-KGDH complex in U251 cells with or without depletion of OGDH, DLST, or DLD using short hairpin RNA (shOGDH, shDLST or shDLD, respectively) was immunoprecipitated with the indicated antibodies and incubated with bacterially purified full-length His–KAT2A protein. d , DLST is required for nuclear distribution of the α-KGDH complex. DLST was depleted from U251 cells, which were reconstituted with expression of RNA interference-resistant (r) wild-type (WT) V5–rDLST or V5–rDLST(R224A/K226E) mutant (V5–rDLST NLS mut). Immunoblotting analyses were performed. Mitochondrial COX IV and IDH2 and nuclear proliferating cell nuclear antigen (PCNA) were used as controls.

    Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.

    Techniques: Immunoprecipitation, shRNA, Incubation, Purification, Expressing, Mutagenesis

    IsoA (4 mg/ml) treatment decreases TGFβ and regulates EMT markers in glioma cells. ( A, B ) IsoA treatment decreased TGFβ mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) IsoA treatment decreases Fibronectin, Vimentin, and increased E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    doi: 10.12659/MSM.910102

    Figure Lengend Snippet: IsoA (4 mg/ml) treatment decreases TGFβ and regulates EMT markers in glioma cells. ( A, B ) IsoA treatment decreased TGFβ mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) IsoA treatment decreases Fibronectin, Vimentin, and increased E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells.

    Article Snippet: U251 cells were transfected by pedue12.4-TGFβ (pTGFβ) using lipofectamine 2000 (Sigma-Aldrich).

    Techniques: Expressing

    IsoA treatment significantly inhibits tumor growth in glioma-bearing mouse model. ( A ) IsoA treatment (4 mg/kg) significantly inhibited tumor growth in the U251-bearing mouse model. ( B ) IsoA treatment decreased TGFβ, Fibronectin, and Vimentin and increases E-cadherin expression levels in tumors as determined by immunohistochemistry.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    doi: 10.12659/MSM.910102

    Figure Lengend Snippet: IsoA treatment significantly inhibits tumor growth in glioma-bearing mouse model. ( A ) IsoA treatment (4 mg/kg) significantly inhibited tumor growth in the U251-bearing mouse model. ( B ) IsoA treatment decreased TGFβ, Fibronectin, and Vimentin and increases E-cadherin expression levels in tumors as determined by immunohistochemistry.

    Article Snippet: U251 cells were transfected by pedue12.4-TGFβ (pTGFβ) using lipofectamine 2000 (Sigma-Aldrich).

    Techniques: Expressing, Immunohistochemistry

    IsoA regulates growth and aggressiveness of glioma cells through TGFβ-induced EMT signal pathway. ( A, B ) TGFβ knockdown (Si-TGFβ) down-regulates Fibronectin and Vimentin and increased E-cadherin mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) TGFβ overexpression (pTGFβ) canceled IsoA-regulated (pTGFβ-IsoA) Fibronectin, Vimentin, and E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells. ( E–G ) TGFβ knockdown (Si-TGFβ) significantly inhibits growth ( E ), migration ( F ), and invasion ( G ) of U251 cells. ( H–J ) TGFβ overexpression abolishes IsoA-inhibited growth ( H ), migration ( I ), and invasion ( J ) of U251 cells

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    doi: 10.12659/MSM.910102

    Figure Lengend Snippet: IsoA regulates growth and aggressiveness of glioma cells through TGFβ-induced EMT signal pathway. ( A, B ) TGFβ knockdown (Si-TGFβ) down-regulates Fibronectin and Vimentin and increased E-cadherin mRNA ( A ) and protein ( B ) expression levels in U251 cells. ( C, D ) TGFβ overexpression (pTGFβ) canceled IsoA-regulated (pTGFβ-IsoA) Fibronectin, Vimentin, and E-cadherin mRNA ( C ) and protein ( D ) expression levels in U251 cells. ( E–G ) TGFβ knockdown (Si-TGFβ) significantly inhibits growth ( E ), migration ( F ), and invasion ( G ) of U251 cells. ( H–J ) TGFβ overexpression abolishes IsoA-inhibited growth ( H ), migration ( I ), and invasion ( J ) of U251 cells

    Article Snippet: U251 cells were transfected by pedue12.4-TGFβ (pTGFβ) using lipofectamine 2000 (Sigma-Aldrich).

    Techniques: Expressing, Over Expression, Migration

    IsoA treatment significantly inhibits growth and aggressiveness of glioma cells. ( A ) IsoA inhibits U251 growth in a dose-depended manner (2, 4, and 6 mg/ml). ( B ) IsoA inhibits U251 growth a time-dependent (24, 48, and 72 h) manner. ( C, D ) IsoA (4 mg/ml) significantly inhibits migration ( C ) and invasion ( D ) of U251 cells after 48-h incubation.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    doi: 10.12659/MSM.910102

    Figure Lengend Snippet: IsoA treatment significantly inhibits growth and aggressiveness of glioma cells. ( A ) IsoA inhibits U251 growth in a dose-depended manner (2, 4, and 6 mg/ml). ( B ) IsoA inhibits U251 growth a time-dependent (24, 48, and 72 h) manner. ( C, D ) IsoA (4 mg/ml) significantly inhibits migration ( C ) and invasion ( D ) of U251 cells after 48-h incubation.

    Article Snippet: U251 cells were transfected by pedue12.4-TGFβ (pTGFβ) using lipofectamine 2000 (Sigma-Aldrich).

    Techniques: Migration, Incubation

    IsoA (4 mg/ml) treatment induces apoptosis of glioma cells. ( A ) IsoA treatment induces apoptosis of U251 cells after 48-h incubation. ( B ) IsoA treatment decreases viability of U251 cells after 48-h incubation. ( C ) IsoA treatment decreases anti-apoptosis gene Bcl-2 and Bcl-w gene expression in U251 cells. ( D ) IsoA treatment increases pro-apoptosis gene caspase-3 and Bad gene expression levels in U251 cells.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Natural Diterpenoid Isoferritin A (IsoA) Inhibits Glioma Cell Growth and Metastasis via Regulating of TGFβ-Induced EMT Signal Pathway

    doi: 10.12659/MSM.910102

    Figure Lengend Snippet: IsoA (4 mg/ml) treatment induces apoptosis of glioma cells. ( A ) IsoA treatment induces apoptosis of U251 cells after 48-h incubation. ( B ) IsoA treatment decreases viability of U251 cells after 48-h incubation. ( C ) IsoA treatment decreases anti-apoptosis gene Bcl-2 and Bcl-w gene expression in U251 cells. ( D ) IsoA treatment increases pro-apoptosis gene caspase-3 and Bad gene expression levels in U251 cells.

    Article Snippet: U251 cells were transfected by pedue12.4-TGFβ (pTGFβ) using lipofectamine 2000 (Sigma-Aldrich).

    Techniques: Incubation, Expressing

    The methylation levels of ZC3H12D in independent clinical samples and the inhibition of ZC3H12D methylation on gene expression. (A) The methylation levels of the CpG site, cg06762457 located in ZC3H12D gene promoter, in 24 clinical blood samples were determined through pyrosequencing. (B) ZC3H12D expression is increased in glioma cell line U251 after treatment with different concentration of demethylation agent 5′-Aza (0, 5, 10, and 20 μM) by western blot. (C) Quantitative analysis of the results from three independent experiments represented in (B) showing that the hypermethylation in ZC3H12D down-regulated gene expression in U251 cell. (D) ZC3H12D expression is increased in breast tumor cell line MCF7 after treatment with different concentration of demethylation agent 5'-Aza (0, 5, 10, and 20 μM) by western blot. (E) Quantitative analysis of the results from three independent experiments represented in (D) showing that the hypermethylation in ZC3H12D inhibited gene expression in MCF7 cell.

    Journal: Frontiers in Aging Neuroscience

    Article Title: DNA Methylation Profiling Reveals the Change of Inflammation-Associated ZC3H12D in Leukoaraiosis

    doi: 10.3389/fnagi.2018.00143

    Figure Lengend Snippet: The methylation levels of ZC3H12D in independent clinical samples and the inhibition of ZC3H12D methylation on gene expression. (A) The methylation levels of the CpG site, cg06762457 located in ZC3H12D gene promoter, in 24 clinical blood samples were determined through pyrosequencing. (B) ZC3H12D expression is increased in glioma cell line U251 after treatment with different concentration of demethylation agent 5′-Aza (0, 5, 10, and 20 μM) by western blot. (C) Quantitative analysis of the results from three independent experiments represented in (B) showing that the hypermethylation in ZC3H12D down-regulated gene expression in U251 cell. (D) ZC3H12D expression is increased in breast tumor cell line MCF7 after treatment with different concentration of demethylation agent 5'-Aza (0, 5, 10, and 20 μM) by western blot. (E) Quantitative analysis of the results from three independent experiments represented in (D) showing that the hypermethylation in ZC3H12D inhibited gene expression in MCF7 cell.

    Article Snippet: For the demethylation test, when cells seeded in 6-well plates reached 40% confluence, U251 and MCF7 were treated with 5′-aza-2′-deoxycytidine (5′-Aza) (Cat. No: A3656-5MG, Sigma, St Louis, MO, USA) with 0, 5, 10, and 20 μM final concentration, a well-used methyltransferase inhibitor, respectively.

    Techniques: Methylation, Inhibition, Expressing, Concentration Assay, Western Blot

    MiR-23a promotes invasion of GBM cells. a The Matrigel invasion assays were performed using six human GBM cell lines: LN443, U373, LN308, U87, U251, and KMG4. * P

    Journal: Signal Transduction and Targeted Therapy

    Article Title: miR-23a promotes invasion of glioblastoma via HOXD10-regulated glial-mesenchymal transition

    doi: 10.1038/s41392-018-0033-6

    Figure Lengend Snippet: MiR-23a promotes invasion of GBM cells. a The Matrigel invasion assays were performed using six human GBM cell lines: LN443, U373, LN308, U87, U251, and KMG4. * P

    Article Snippet: U87 cells (ATCC#HTB-14) and U251 cells (ATCC#CRL2219) were purchased from the American Type Culture Collection (ATCC).

    Techniques: