fluorescent hat assay kit  (Active Motif)

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

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

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