primary antibodies against ezh2  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: ( a ) Western blot of EZH2, V-5 tag, H3K27me3, and H3 total for untransfected, empty vector (EV), wild-type (WT) EZH2, EZH2 Y641F , EZH2 A677G , and EZH2 H689A/F667I with α-GAPDH as loading control. ( b ) Summary of EpiProfile analysis of K27 methylation for histone H3 variants across EZH2 mutants. Error bars represent 1 standard deviation and asterisk represents statistical significance (q-value < 0.05). ( c ) Principal component analysis on replicates of acquired multi-omics datasets.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques: Western Blot, Plasmid Preparation, Control, Methylation, Standard Deviation, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: Normalized genomic distributions of ( a ) H3K27me3 CUT&Tag enriched peaks; and ( b ) Open chromatin regions in EZH2 GOF mutants. Profile plot ( c ) and heatmaps ( d ) of CUT&Tag H3K27me3 and ATAC-Seq peaks ± 5 kb from the centers of putative EZH2 targets across EZH2 mutants.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: ( a ) Gene browser tracks showing CUT&Tag H3K27me3, ATAC-Seq, and RNA-Seq readouts over WNT16 , RPRM and EGFR genes. ( b ) Gene ontology and ( c ) KEGG pathways of EZH2 target genes in EZH2 GOF mutants.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques: RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: String analysis of protein–protein networks for dysregulated proteins in ( a ) EZH2 GOF mutants and ( b ) EZH2 LOF.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: Heatmap showing top 40 differential metabolites across each EZH2 mutant.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques: Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures

doi: 10.3390/ijms241411378

Figure Lengend Snippet: ( a ) Boxplots for NAD+ and ATP quantification via colorimetric assay and ( b ) expression heatmap for selected genes belonging to NAD+/NADH biochemical pathway in EZH2 mutants.

Article Snippet: The membrane was blocked with TBS Blocking Buffer (Li-Cor, 927-60001, Lincoln, NE, USA) for an hour at room temperature and then incubated overnight at 4 °C with primary antibodies against EZH2 (Cell Signaling Technology, 5246), H3 (Abcam, ab1791, Cambridge, UK), H3K27me3 (Cell Signaling Technology, 9733), GAPDH (Cell Signaling Technology, 2118S) and V5 (Invitrogen, R960-25).

Techniques: Colorimetric Assay, Expressing

Journal: Cell reports

Article Title: Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor

doi: 10.1016/j.celrep.2022.111363

Figure Lengend Snippet: (A) Doxycycline (Dox)-induced reexpression of SUZ12 in MPNST cells restored trimethylated histone H3 lysine 27 (H3K27me3), as shown by western blotting. (B) Network plot demonstrating the connection of three significantly enriched pathways of the differentially expressed genes (DEGs) caused by PRC2 restoration. DEGs were derived from a combined analysis of the RNA-seq results obtained from all three PRC2-null MPNST cell lines, comparing samples with induced expression of SUZ12 to controls; p adj < 0.05, ∣log2 fold change∣ > 1. (C) Gene set enrichment plot shows that PRC2 restoration significantly decreased genes in the “cell-fate commitment” pathway. NES, normalized enrichment score. (D) Venn diagram showing the number of overlapped H3K27me3 peaks in three MPNST cell lines upon PRC2 restoration. (E) PRC2 restoration drove the genome-wide gain of H3K27me3 occupancy as demonstrated by overlapped ChIP peaks in MPNST cells. (F) Pie chart showing the distribution of transcriptional alterations among the 5,816 genes associated with H3K27me3 peaks found to be common in at least two cell lines upon Dox induction. Dark gray, genes not expressed; light gray, genes expressed but not changed; blue, significantly downregulated; red, significantly upregulated; p adj < 0.05. (G) Bar plot shows the enrichment of bivalent genes among the 695 downregulated genes upon PRC2 restoration. (H) Heatmap of ChIP-seq signals shows that PRC2 restoration caused gain of H3K27me3 and loss of H3K27ac at 850 common bivalent sites in MPNST cells.

Article Snippet: Primary antibodies against FOXC1, EZH2, SUZ12 and IgG isotype control were incubated overnight with Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Invitrogen) followed with washing using Washing Buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.005 Triton X-100, 1 mM EDTA, de-ionized H 2 O).

Techniques: Western Blot, Derivative Assay, RNA Sequencing Assay, Expressing, Genome Wide, ChIP-sequencing

Journal: Cell reports

Article Title: Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor

doi: 10.1016/j.celrep.2022.111363

Figure Lengend Snippet: (A) Heatmap showing the median percentage of reduction in proliferation when a candidate core TF was knocked down using three distinct siRNAs, in comparison with the control siRNA. Data are shown in the order of average median reduction from the highest to the lowest in four PRC2-deficient and two PRC2-WT cell lines. Data summarize two replicates per siRNA per cell line. Direct PRC2 targets are highlighted in red. (B) Western blot shows the reduction of protein levels of FOXC1 and HOXB8 caused by Dox-induced SUZ12 reexpression. (C) Example integrative genomics viewer (IGV) tracks around the FOXC1 (top) and HOXB8 (bottom) loci demonstrate the gain of H3K27me3, loss of super enhancer (SE), and transcriptional reduction accompanying the Dox-induced PRC2 restoration in T265 cells. (D) Cell growth assay using individual siRNAs targeting FOXC1 in T265 (first panel) and ST88-14 (second panel). siC, siControl; siF#1, siFOXC1 #1; and siF#5, siFOXC1 #5. Quantification (third panel) and representative picture (fourth panel) of colony formation when FOXC1 was knocked out in T265 cells by CRISPR-Cas9 are shown. (E) Cell growth assay using individual siRNAs targeting HOXB8 in T265 (first panel) and ST88-14 (second panel). siH#4, siHOXB8 #4, and siH#6, siHOXB8 #6. Quantification (third panel) and representative picture (fourth panel) of colony formation when HOXB8 was knocked out in T265 cells by CRISPR-Cas9 are shown. The average of five replicates is presented; mean ± SEM; scale bar, 1 mm.

Article Snippet: Primary antibodies against FOXC1, EZH2, SUZ12 and IgG isotype control were incubated overnight with Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Invitrogen) followed with washing using Washing Buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.005 Triton X-100, 1 mM EDTA, de-ionized H 2 O).

Techniques: Western Blot, Growth Assay, CRISPR

Journal: Cell reports

Article Title: Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor

doi: 10.1016/j.celrep.2022.111363

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies against FOXC1, EZH2, SUZ12 and IgG isotype control were incubated overnight with Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Invitrogen) followed with washing using Washing Buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.005 Triton X-100, 1 mM EDTA, de-ionized H 2 O).

Techniques: Recombinant, Lysis, Protease Inhibitor, Western Blot, Bradford Assay, Magnetic Beads, Multiplex Assay, Next-Generation Sequencing, Generated, Sequencing, Negative Control, Plasmid Preparation, Software, Microscopy

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Transcriptome analyses reveals Ezh2 is a potential regulator of age-related LA fibrosis. Total RNA were extracted from mice LA tissues and reversely transcribed to cDNA for RNA-Seq. ( A ) Volcano plots revealed that 945 of DEGs were up-regulated (log 2 fold change >0.7; p <0.05; red dots) and 921 down-regulated (log 2 fold change<0.7; p <0.05; blue dots) in aged vs young LA. ( B ) Heatmap summaries. ( C ) GO term enrichment analyses. The vertical coordinates are the terms (the biological process in green; the cellular component in Orange; the molecular function in blue) and the horizontal coordinates are the numbers of DEGs. ( D ) Venn diagram showing the overlap of GO terms as indicated, and Ezh2 gene was chosen. ( E and F ) Down-regulated level of Ezh2 was validated by WB of either mice LA tissues ( E ) or isolated pure AFs ( F ). Representative WB images of Ezh2 and their densitometry analyses. n=6 mice. Data are presented as mean ± SD. # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: RNA Sequencing, Isolation

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Ezh2 is down-regulated in a RS model of AFs. ( A ) Schematic diagram of our RS model of AFs isolated from young mice and classified as P0, P1 and P2. bar=20μm. P0, P1 and P2 cells were harvested for multiple analyses in B~G. ( B and C ) Representative images of SA-β-gal staining and their quantitative analyses (n=6 mice in each group). ( D ) Transcriptions of Ezh2 was assessed by qRT-PCR (n=3 in each group). ( E ) Representative WB images of Ezh2 and CDKIs markers as indicated, as well as their densitometry analyses (n=6). ( F and G ) Transcriptions of CDKIs ( F ) and SASP genes (IL-1α, IL-1β, IL-6, MCP-1; ( G ) were evaluated by qRT-PCR (n=3 in each group). Data are presented as mean ± SD. * p <0.05, ** p <0.01, # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: Isolation, Staining, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Knockdown of Ezh2 induces RS in the early passage of AFs. ( A ) Schematic diagram of Ezh2 silencing in AFs. Isolated, plated and stably attached P1 AFs on Day-1 were transfected with scramble (Scr) or Ezh2 siRNAs (100nM) for 72h and harvested on Day-4 for analyses. ( B ) Representative WB images showing the efficiency of Ezh2 knocking-down ( left ), as well as its densitometry analyses ( right ; n=6). Si-Ezh2-2 was chosen to be used in the thereafter experiments. ( C ) Transcriptions of Ezh2 after transfection of si-Ezh2-2 was assessed by qRT-PCR (n=3 in each group). ( D ) Representative images of SA-β-gal staining. SA-β-gal activity was quantitated and expressed as percent of the positive cells (n=6). bar=100μm. ( E ) Representative WB images of Ezh2 and CDKIs, as well as their densitometry analyses (n=6). ( F and G ) Transcriptions of CDKIs ( F ) and SASP genes ( G ) were evaluated by qRT-PCR (n=3). Data are presented as mean ± SD. * p <0.05, ** p <0.01, # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: Knockdown, Isolation, Stable Transfection, Transfection, Quantitative RT-PCR, Staining, Activity Assay

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Overexpression of Ezh2 alleviates RS in the late passage of AFs. ( A ) Schematic diagram of Ezh2 overexpression in P2 AFs. AFs on Day-6 were transduced with Adv-Ezh2 and harvested 72h later for analyses. ( B ) Transcriptions of Ezh2 was assessed by qRT-PCR (n=3 in each group). ( C ) Representative WB images of Ezh2 and CDKIs, as well as their densitometry quantitative analyses (n=6). ( D ) Representative images of SA-β-gal staining, SA-β-gal activity was quantified and expressed as percentage of the positive cells (n=6). bar=100μm. ( E and F ) Transcriptions of CDKIs ( E ) and SASP genes ( F ) were evaluated by qRT-PCR (n=3). Data are presented as mean ± SD. * p <0.05, ** p <0.01, # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: Over Expression, Transduction, Quantitative RT-PCR, Staining, Activity Assay

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Changes in H3K27me3 Levels in our RS models ( A ), after Knockdown of Ezh2 ( B and C ), and Overexpression of Ezh2 ( D and E ). ( A ) Representative WB images of H3K27me3 using SDS-protein samples of above and its densitometry analyses (n=6). ( B ) Representative WB images of H3K27me3 using SDS-protein samples of above and its densitometry analyses (n=6). ( C ) Representative immunofluorescence images of H3K27me3 (green) expression in cells manipulated as above . bar=100μm. ( D ) Representative WB images of H3K27me3 using SDS-protein samples of above and its densitometry analyses (n=6). ( E ) Representative immunofluorescence images of H3K27me3 (green) expression in cells manipulated as above . bar=100μm. Data are presented as mean ± SD. # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: Knockdown, Over Expression, Immunofluorescence, Expressing

Journal: Journal of Inflammation Research

Article Title: Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes

doi: 10.2147/JIR.S374951

Figure Lengend Snippet: Inhibition of Ezh2ʹs methyltransferase activities promotes RS in the early passage of AFs. ( A ) Schematic diagram of Ezh2 inhibition in P1 AFs. AFs on Day-1 were treated with GSK-126 (1μM, 72h) or GSK-343 (1μM, 72h) and harvested on Day-4 for the following analyses. ( B ) Representative WB images of Ezh2, H3K27me3 and CDKIs, as well as their densitometry quantitative analyses (n=6). ( C ) Representative immunofluorescence images of H3K27me3 expression (green). bar=100μm. ( D ) Representative images of SA-β-gal staining and SA-β-gal activity was quantitated and expressed as a percentage of SA-β-gal-positive cells (n=6). bar=100μm. ( E and F ) Transcriptions of CDKIs ( E ) and SASP genes ( F ) were evaluated by qRT-PCR (n=3). Data are presented as mean ± SD. * p <0.05, ** p <0.01, # p <0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST (Tris-HCl buffered saline supplemented with 0.05% Tween-20) for 2 h at room temperature and then incubated with primary antibodies against Ezh2 (Abclonal, A11085), H3K27me3 (CST, #9733S), Timp1 (Abcam, ab179580), Timp2 (Abcam, ab180630), Timp3 (Abcam, ab39184), Timp4 (Abclonal, A6416), MMP1 (Abcam, ab137332), MMP8 (Abcam, ab53017), MMP13 (Abcam, ab39012), p16 (Abcam, ab211542), p19 (Abcam, ab80), p21 (Abcam, ab188224), collagen-I (Abcam, ab254113), collagen-III (Servicebio, GB111323), or GAPDH (Servicebio, GB11002) at 4°C overnight.

Techniques: Inhibition, Immunofluorescence, Expressing, Staining, Activity Assay, Quantitative RT-PCR