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fluorescent hat assay kit  (Active Motif)


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    Active Motif fluorescent hat assay kit
    a Subcellular localization of HWS1 and HWS2 fused with the yellow <t>fluorescent</t> protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b <t>HAT</t> activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.
    Fluorescent Hat Assay Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/fluorescent+hat+assay+kit/fluorescent+hat+assay+kit/pmc10837208-300-14-18
    Average 90 stars, based on 1 article reviews
    fluorescent hat assay kit - by Bioz Stars, 2026-11
    90/100 stars

    Images

    1) Product Images from "Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier"

    Article Title: Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

    Journal: Nature Communications

    doi: 10.1038/s41467-024-45377-x

    a Subcellular localization of HWS1 and HWS2 fused with the yellow fluorescent protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b HAT activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.
    Figure Legend Snippet: a Subcellular localization of HWS1 and HWS2 fused with the yellow fluorescent protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b HAT activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.

    Techniques Used: Marker, Activity Assay, Fluorescence, Western Blot, RNA Sequencing, Expressing, ChIP-sequencing, Biomarker Discovery, Ubiquitin Proteomics, ChIP-qPCR

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    other:

    Article Title: Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails
    Article Snippet: Fluorescence HAT assay HAT reactions were performed for 17 hours at 25 °C using 20 pmol of nucleosomes, hFACT proteins, and the mixed samples in the presence of 2 pmol of human Gcn5 catalytic domain (Enzo Life Sciences) and 2.5 nmol of Acetyl-CoA in 20 mM Tris-HCl pH 7.0 in a total volume of 50 μl.

    Article Title: NAT10 modulators for treating or preventing laminopathies, aging and cancer
    Article Snippet: DNA was purified using the Qiagen PCR purification kit and 1500 ng of DNA was analysed on a 1.5% agarose gel.

    Article Title: Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.
    Article Snippet: Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties.. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi:10.1007/s12035015-9314-z, 2015).. This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal.

    In Vitro:

    Article Title: FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.
    Article Snippet: .. In vitro acetylation assay In vitro acetylation assay was performed following instructions provided by Fluorescent HAT Assay Kit (Active Motif, 56100). ..

    Article Title: Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier
    Article Snippet: .. For the in vitro HAT assay, the intrinsic HAT activity was detected using the fluorescent HAT Assay Kit (Active Motif) following the manufacturer’s instructions with some modifications. .. In brief, the purified proteins (MBP-HWS2, MBP, and positive control p300) were incubated with 20 μl histone H4 peptide in a 50 μl reaction mix that included 6 μl of 5× HAT assay buffer and 5 μl of acetyl-CoA (0.5 mM) at 28°C for 1 h. The corresponding fluorescence signals were detected using a spectrophotometer with excitation at 375 nm and emission at 460 nm (Thermo Scientific, Varioskan LUX).

    Acetylation Assay:

    Article Title: FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.
    Article Snippet: .. In vitro acetylation assay In vitro acetylation assay was performed following instructions provided by Fluorescent HAT Assay Kit (Active Motif, 56100). ..

    HAT Assay:

    Article Title: FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.
    Article Snippet: .. In vitro acetylation assay In vitro acetylation assay was performed following instructions provided by Fluorescent HAT Assay Kit (Active Motif, 56100). ..

    Article Title: Methods of increasing the cytotoxicity of chemotherapeutic agents with multisubstrate inhibitors of histone, protein-lys, polyamine acetylation, and polyamine metabolism
    Article Snippet: .. Assays were performed using the fluorescent HAT assay kit (Active Motif, Carlsbad, Calif.), an H3 peptide as substrate (residues 5-23), the p300 catalytic domain as enzyme, and 50 μM inhibitor in duplicate, according to the manufacturer's instructions. ..

    Article Title: Soluble fibre supplementation with and without a probiotic in adults with asthma: A 7-day randomised, double blind, three way cross-over trial
    Article Snippet: .. A deacetylated HDAC standard curve will be used to quantify HDAC activity (uM/ug). (d) Total HAT enzyme activity assay Measurement of total HAT enzyme activity in PBMC nuclear protein extracts will be performed using the Active Motif fluorescent HAT Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. ..

    Article Title: Soluble fibre supplementation with and without a probiotic in adults with asthma: A 7-day randomised, double blind, three way cross-over trial
    Article Snippet: .. Measurement of HAT enzyme activity in PBMC nuclear protein extracts was performed using the Active Motif fluorescent HAT Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. .. Fluorescence was measured at 360 nm excitation and 460 nm emission (FLUOstar Optima, BMG Labtech, Durham, NC, USA).

    Article Title: Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier
    Article Snippet: .. For the in vitro HAT assay, the intrinsic HAT activity was detected using the fluorescent HAT Assay Kit (Active Motif) following the manufacturer’s instructions with some modifications. .. In brief, the purified proteins (MBP-HWS2, MBP, and positive control p300) were incubated with 20 μl histone H4 peptide in a 50 μl reaction mix that included 6 μl of 5× HAT assay buffer and 5 μl of acetyl-CoA (0.5 mM) at 28°C for 1 h. The corresponding fluorescence signals were detected using a spectrophotometer with excitation at 375 nm and emission at 460 nm (Thermo Scientific, Varioskan LUX).

    Activity Assay:

    Article Title: Soluble fibre supplementation with and without a probiotic in adults with asthma: A 7-day randomised, double blind, three way cross-over trial
    Article Snippet: .. A deacetylated HDAC standard curve will be used to quantify HDAC activity (uM/ug). (d) Total HAT enzyme activity assay Measurement of total HAT enzyme activity in PBMC nuclear protein extracts will be performed using the Active Motif fluorescent HAT Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. ..

    Article Title: Soluble fibre supplementation with and without a probiotic in adults with asthma: A 7-day randomised, double blind, three way cross-over trial
    Article Snippet: .. Measurement of HAT enzyme activity in PBMC nuclear protein extracts was performed using the Active Motif fluorescent HAT Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. .. Fluorescence was measured at 360 nm excitation and 460 nm emission (FLUOstar Optima, BMG Labtech, Durham, NC, USA).

    Article Title: Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier
    Article Snippet: .. For the in vitro HAT assay, the intrinsic HAT activity was detected using the fluorescent HAT Assay Kit (Active Motif) following the manufacturer’s instructions with some modifications. .. In brief, the purified proteins (MBP-HWS2, MBP, and positive control p300) were incubated with 20 μl histone H4 peptide in a 50 μl reaction mix that included 6 μl of 5× HAT assay buffer and 5 μl of acetyl-CoA (0.5 mM) at 28°C for 1 h. The corresponding fluorescence signals were detected using a spectrophotometer with excitation at 375 nm and emission at 460 nm (Thermo Scientific, Varioskan LUX).

    Enzyme Activity Assay:

    Article Title: Soluble fibre supplementation with and without a probiotic in adults with asthma: A 7-day randomised, double blind, three way cross-over trial
    Article Snippet: .. A deacetylated HDAC standard curve will be used to quantify HDAC activity (uM/ug). (d) Total HAT enzyme activity assay Measurement of total HAT enzyme activity in PBMC nuclear protein extracts will be performed using the Active Motif fluorescent HAT Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. ..



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    a Subcellular localization of HWS1 and HWS2 fused with the yellow <t>fluorescent</t> protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b <t>HAT</t> activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.
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    a Subcellular localization of HWS1 and HWS2 fused with the yellow <t>fluorescent</t> protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b <t>HAT</t> activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.
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    Image Search Results


    a Subcellular localization of HWS1 and HWS2 fused with the yellow fluorescent protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b HAT activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.

    Journal: Nature Communications

    Article Title: Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

    doi: 10.1038/s41467-024-45377-x

    Figure Lengend Snippet: a Subcellular localization of HWS1 and HWS2 fused with the yellow fluorescent protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b HAT activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL- hws1 WYJ7 /hws2 CG14 ; Lane #2: NIL- HWS1 CG14 / hws2 CG14 ; Lane #3: NIL- hws1 WYJ7 /HWS2 WYJ7 ; and Lane #4: NIL- HWS1 CG14 / HWS2 WYJ7 . d The distribution profile of averaged H4Ac occupancy across the gene body in NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 (**** p < 0.0001, calculated by two-sided Welch’s t -test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e . All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase ( LOC_Os08g23810 and LOC_Os09g10600 ) and OsKASI ( LOC_Os06g09630 ) in the young panicles of NIL- HWS1 CG14 /hws2 CG14 and NIL- hws1 WYJ7 /hws2 CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL- HWS1 CG14 /hws2 CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h . j , k , Measurement of biotin level in NIL- hws1 WYJ7 /hws2 CG14 versus NIL- HWS1 CG14 / hws2 CG14 plants ( j ) and NIL- hws1 WYJ7 /hws2 CG14 versus NIL- hws1 WYJ7 /HWS2 WYJ7 plants ( k ). Data in b , h , i , j , and k are mean ± SEM ( n = 3 ( b , h , and i ) or 4 ( j and k ) biological replicates). In b , different letters denote significant differences ( p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t -test was used to generate the p -values in h – k . The experiments in a and c were repeated three times independently, with similar results.

    Article Snippet: For the in vitro HAT assay, the intrinsic HAT activity was detected using the fluorescent HAT Assay Kit (Active Motif) following the manufacturer’s instructions with some modifications.

    Techniques: Marker, Activity Assay, Fluorescence, Western Blot, RNA Sequencing, Expressing, ChIP-sequencing, Biomarker Discovery, Ubiquitin Proteomics, ChIP-qPCR

    HAT inhibitor CPTH2 decreases cell viability in tumor cells. a In vitro HAT activity was measured in nuclear extracts of Thyroid papillary K1 (gray) and clear cell renal carcinoma ccRCC 786-O (dark gray) cell lines treated with CPTH2 (100 μM) for 24 and 48 h, respectively, and compared to untreated and DMSO samples. b Cell proliferation measured in K1 (gray) and 786-O (dark gray) cell lines in DMSO or during CPTH2 treatment at increased times (h) showed strong reduction at 48 h. c FACS analysis of 786-O cell line grown in DMSO and treated for 48 h with CPTH2. d Apoptotic FACS profile and percentage of apoptotic cells strongly enhanced after 48 h CPTH2 treatment in both K1 and 786-O cell lines

    Journal: Clinical Epigenetics

    Article Title: KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

    doi: 10.1186/s13148-018-0473-4

    Figure Lengend Snippet: HAT inhibitor CPTH2 decreases cell viability in tumor cells. a In vitro HAT activity was measured in nuclear extracts of Thyroid papillary K1 (gray) and clear cell renal carcinoma ccRCC 786-O (dark gray) cell lines treated with CPTH2 (100 μM) for 24 and 48 h, respectively, and compared to untreated and DMSO samples. b Cell proliferation measured in K1 (gray) and 786-O (dark gray) cell lines in DMSO or during CPTH2 treatment at increased times (h) showed strong reduction at 48 h. c FACS analysis of 786-O cell line grown in DMSO and treated for 48 h with CPTH2. d Apoptotic FACS profile and percentage of apoptotic cells strongly enhanced after 48 h CPTH2 treatment in both K1 and 786-O cell lines

    Article Snippet: Histone acetyltransferase activity was measured with fluorescent in vitro HAT Assay Kit (Active Motif, CA) in nuclear extracts (7 μg) prepared with EpiQuikTM Nuclear Extraction Kit I (Epigentek, NY) treated 24 or 48 h with CPTH2 100 μM or solvent DMSO.

    Techniques: In Vitro, Activity Assay

    CPTH2 preferentially inhibits HAT-p300 in ccRCC 786-O cells. a In vitro HAT assay was performed on recombinant p300 (red), GCN5 (green), and PCAF (blue). Control ( c ) in presence of HAT inhibitor anacardic acid, AA (15 μM), and CPTH2 (400 and 600 μM). CPTH2 shows higher selectivity in the inhibition of p300. b Prolonged incubation with CPTH2 does not affect mRNA expression of p300. c treatment of ccRCC 786-O with CPTH2 (100 μM) at indicated times clears the immunostaining of p300 with respect to untreated and DMSO cells. d Silencing of p300 in 786-O cells caused decrease of p300-mRNA expression (light gray) and lowering of cell viability (gray) with respect to nc control. e Cytoskeleton of 786-O cells silenced for p300 (18 h) was stained with phalloidine. It is heavily modified and fully comparable to the cytoskeleton of cells treated with CPTH2 shown in Fig. , and histogram with percentage of stress fibers/cell found after 24 h p300 si (light gray) compared to control, nc (dark gray). f Histograms reporting the number of proliferating cells, percentage of stress fiber/cell and number of migrating cells in, respectively, nc control (black) and p300 si cells (gray) grown in DMSO and in CPTH2 for 48 h

    Journal: Clinical Epigenetics

    Article Title: KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

    doi: 10.1186/s13148-018-0473-4

    Figure Lengend Snippet: CPTH2 preferentially inhibits HAT-p300 in ccRCC 786-O cells. a In vitro HAT assay was performed on recombinant p300 (red), GCN5 (green), and PCAF (blue). Control ( c ) in presence of HAT inhibitor anacardic acid, AA (15 μM), and CPTH2 (400 and 600 μM). CPTH2 shows higher selectivity in the inhibition of p300. b Prolonged incubation with CPTH2 does not affect mRNA expression of p300. c treatment of ccRCC 786-O with CPTH2 (100 μM) at indicated times clears the immunostaining of p300 with respect to untreated and DMSO cells. d Silencing of p300 in 786-O cells caused decrease of p300-mRNA expression (light gray) and lowering of cell viability (gray) with respect to nc control. e Cytoskeleton of 786-O cells silenced for p300 (18 h) was stained with phalloidine. It is heavily modified and fully comparable to the cytoskeleton of cells treated with CPTH2 shown in Fig. , and histogram with percentage of stress fibers/cell found after 24 h p300 si (light gray) compared to control, nc (dark gray). f Histograms reporting the number of proliferating cells, percentage of stress fiber/cell and number of migrating cells in, respectively, nc control (black) and p300 si cells (gray) grown in DMSO and in CPTH2 for 48 h

    Article Snippet: Histone acetyltransferase activity was measured with fluorescent in vitro HAT Assay Kit (Active Motif, CA) in nuclear extracts (7 μg) prepared with EpiQuikTM Nuclear Extraction Kit I (Epigentek, NY) treated 24 or 48 h with CPTH2 100 μM or solvent DMSO.

    Techniques: In Vitro, HAT Assay, Recombinant, Control, Inhibition, Incubation, Expressing, Immunostaining, Staining, Modification