Journal: bioRxiv

Article Title: Triptolide ameliorates renal tubulointerstitial fibrosis through EZH2

doi: 10.1101/2023.01.29.526092

Figure Lengend Snippet: A and B . The expression of EZH2 in sham or UUO kidneys was analyzed by Western blotting and further quantified. C and D . HK2 human renal epithelial cells were starved overnight, then stimulated with TGF-β and treated with various concentrations of triptolide for 48h. The expression of EZH2 was analyzed by Western blotting and further quantified. One representative of at least three independent experiments is shown. Data represents the mean◻±◻SD. NS represents not significant. *** p ◻<◻0.001.

Article Snippet: Unspecific binding on the PVDF membrane was blocked by incubation with the blocking buffer (5% non-fat milk, 20mM Tris-HCl, 150mM NaCl, PH=8.0, 0.01%Tween 20) for 1 hour at room temperature, which was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam), anti-Collagen I (1:500, sc-293182, Santa Cruz), anti-α-SMA (1:1000, ET1607-53, HUABIO), anti-EZH2 (1:1000, 5246S, Cell Signaling Technology) antibody, anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-Vimentin (1:1000, R1308-6, HUABIO), anti-Snail(1:1000, A11794, ABclonal), anti-pSmad3 (1:1000, ET1609-41, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech) antibodies overnight at 4◻.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Triptolide ameliorates renal tubulointerstitial fibrosis through EZH2

doi: 10.1101/2023.01.29.526092

Figure Lengend Snippet: HK2 human renal epithelial cells were starved overnight and followed by stimulation with TGF-β and treatment with 20 μM of 3-DZNeP and 100nM of triptolide for 48h. Cell lysates were extracted. A and B . The expression of EZH2 was analyzed by Western blotting and further quantified. C-E . The expression of fibronectin and collagen I were analyzed by Western blotting and then quantified. F and G . The expression of N-cadherin, vimentin and snail were analyzed by Western blotting and then quantified. H and I . The expression of phosphorylated Smad3 (pSmad3) was analyzed by Western blotting and then quantified. One representative of at least three independent experiments is shown. Data represents the mean◻±◻SD. NS represents not significant.* p ◻<◻0.05. ** p ◻<◻0.01. *** p ◻<◻0.001.

Article Snippet: Unspecific binding on the PVDF membrane was blocked by incubation with the blocking buffer (5% non-fat milk, 20mM Tris-HCl, 150mM NaCl, PH=8.0, 0.01%Tween 20) for 1 hour at room temperature, which was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam), anti-Collagen I (1:500, sc-293182, Santa Cruz), anti-α-SMA (1:1000, ET1607-53, HUABIO), anti-EZH2 (1:1000, 5246S, Cell Signaling Technology) antibody, anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-Vimentin (1:1000, R1308-6, HUABIO), anti-Snail(1:1000, A11794, ABclonal), anti-pSmad3 (1:1000, ET1609-41, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech) antibodies overnight at 4◻.

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer

doi: 10.3390/cancers15051569

Figure Lengend Snippet: Applied antibodies used in the immunohistochemical staining of three used BTC cell lines KKU-055, NOZ and OCUG-1.

Article Snippet: EZH2 , Cell Signaling , 5246S , D2C9 , High pH , rtu , Ultraview , Ventana.

Techniques: Immunohistochemical staining, Staining, Incubation

Journal: Cell Death & Disease

Article Title: Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy

doi: 10.1038/s41419-023-05668-5

Figure Lengend Snippet: A Subcellular localization analysis suggested that RNF157-AS1 was localized mainly in the nucleus in EOC cells. B The RPISeq database showed that RNF157-AS1 had a very high interaction predicting score with HMGA1 protein (RF:0.8; SVM:0.88). C RNF157-AS1 expression had a positive correlation with HMGA1 expression ( R = 0.32, P = 2.5*10 −13 ) according to the GEPIA database. D Western blot analysis showed that RNF157-AS1 pulled down a greater amount of protein than did the control. E The RIP assay suggested HMGA1 interacted with RNF157-AS1 in not only RNF157-AS1 overexpression but also control EOC cells. F According to the RPISeq database, EZH2 had the highest interaction predicting score with RNF157-AS1 among the four subunits of the PRC2 complex. G EZH2 had a similar interaction predicting scores between RNF157-AS1 and HOTAIR; H RNF157-AS1 had a positive correlation with HMGA1 ( R = 0.25, P = 5.6*10 −9 ) according to the GEPIA database. I Western blot analysis showed that EZH2 was present only in the RNF157-AS1 (sense) precipitate compared with the control (antisense) precipitate. J The RIP assay suggested EZH2 interacted with RNF157-AS1 in not only RNF157-AS1 overexpressing but also control EOC cells. Data were obtained from at least three independent experiments. *** P < 0.001 ( t -test).

Article Snippet: Anti-EZH2(5246S), anti-LC3B (3868S), and anti-β-actin (4970S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy

doi: 10.1038/s41419-023-05668-5

Figure Lengend Snippet: A Differentially expressed mRNAs after RNF157-AS1 knockdown with hierarchical clustering. B Differentially expressed mRNAs shown on a volcano plot. C and D RNF157-AS1 expression had a negative correlation with DIRAS3 expression ( R = −0.39, P = 1.2*10 −19 ) and ULK1 expression ( R = −0.3, P = 8.4*10 −12 ). E–G The mRNA and protein expression level of DIRAS3 after RNF157-AS1 knockdown or overexpression. Full-length blots are presented in Supplementary Figs. S and S . H–J The mRNA and protein expression level of ULK1 after RNF157-AS1 knockdown or overexpression. Full-length blots are presented in Supplementary Figs. S and S . K and L ChIP-qPCR assay demonstrated that inhibiting RNF157-AS1 suppressed the binding of EZH2 to the promoter of the DIRAS3 genes and that RNF157-AS1 overexpression enhanced the binding of EZH2 to the promoter of the DIRAS3. M and N The ChIP assay demonstrated that diminishing of RNF157-AS1 decreased the binding of HMGA1 to the promoter of ULK1 genes and RNF157-AS1 overexpression enhanced the binding of HMGA1 to the promoter of ULK1. Data were obtained from at least three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 ( t -test).

Article Snippet: Anti-EZH2(5246S), anti-LC3B (3868S), and anti-β-actin (4970S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Over Expression, Binding Assay

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Western blots of EZH2 immunoprecipitations (IP) performed using nuclear lysates prepared from SW480 cells untreated or treated with 250 μM H 2 O 2 for 30 minutes. IgG IP serves as a negative control. Input is nuclear lysates used for IP. B . EZH2 IP performed using nuclear lysates prepared from human colorectal cancer organoids treated as in A. C . β-catenin IP performed using nuclear lysates prepared from EZH2 knockdown (KD) and empty vector (EV) SW480 cells treated as in A. D . β-catenin IP performed using nuclear lysates prepared from PTEN KD and EV SW480 cells treated with 2 μM EZH2 inhibitor (EZH2i, GSK-503) or DMSO for 72 hours. E . β-catenin IP preformed using nuclear lysates prepared from SW480 cells untreated or treated with 10 μM AKT inhibitor (AKTi, GSK-690693) for 48 hours followed by no additional treatment or co-treatment with 250 μM H 2 O 2 for 30 minutes. Whole cell extract (WCE) is total protein prior to cellular fractionation. F . HA IP performed using nuclear lysates prepared from HA-EZH2 wildtype (WT) and HA-EZH2 phospho-null (PN) S21A expressing SW480 cells treated as in A.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: Western Blot, Negative Control, Plasmid Preparation, Cell Fractionation, Expressing

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Western blots of Flag immunoprecipitations (IP) performed using nuclear lysates prepared from Flag β-catenin wildtype (WT), Flag β-catenin K19R, Flag β-catenin K49R and Flag β-catenin K19/49R expressing SW480 cells treated with 250 μM H 2 O 2 for 30 minutes and Flag β-catenin WT expressing untreated cells. Input is nuclear lysates used for IP. IP with beads only serves as a negative control. B . Trimethyl lysine (Me3-K) IP performed using nuclear lysates prepared from Flag β-catenin WT and Flag β-catenin K49R expressing SW480 cells treated as in A. IgG IP serves as a negative control. C. Flag IP performed using nuclear lysates prepared from Flag-tagged β-catenin WT and Flag-tagged β-catenin K49R expressing PTEN KD or empty vector (EV) SW480 cells. D . Flag IP performed using nuclear lysates prepared from Flag-tagged β-catenin WT expressing SW480 cells treated with 2 μM EZH2 inhibitor (EZH2i, GSK-503) or DMSO for 72 hours and with or without co-treatment with 250 μM H 2 O 2 for 30 minutes. Cells not expressing Flag plasmid were used as a negative control for the IP. E . β-catenin IP preformed using nuclear lysates prepared from EV or PTEN KD SW480 cells treated with EZH2 inhibitor as in D.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: Western Blot, Expressing, Negative Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Western blots of TCF1 immunoprecipitation (IP) performed using nuclear lysates prepared from Flag-tagged β-catenin wildtype (WT) expressing SW480 cells treated with 1 μM EZH2 inhibitor (EZH2i, GSK-503) or DMSO for 72 hours with or without co-treatment with 250 μM H 2 O 2 for 30 minutes. IgG IP serves as a negative control. Input is nuclear lysates used for IP. B . Flag IP performed using nuclear lysates prepared from Flag-tagged β-catenin WT expressing SW480 cells treated as in A. IPs were performed using the same nuclear lysate in A. C . β-catenin IP performed using nuclear lysates prepared from PTEN knockdown (KD) or empty vector (EV) SW480 cells treated with 2 μM EZH2 inhibitor (EZH2i, GSK-503) or DMSO for 72 hours. D . Flag IP performed using nuclear lysates prepared from Flag-tagged β-catenin WT and Flag-tagged β-catenin K49R expressing SW480 cells untreated or treated with 250 μM H 2 O 2 for 30 minutes. IP with beads only serves as a negative control. E . Flag IP performed using nuclear lysates prepared from Flag-tagged β-catenin WT and Flag-tagged β-catenin K49R expressing PTEN KD or EV SW480 cells.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: Western Blot, Immunoprecipitation, Expressing, Negative Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Western blots of chromatin lysate prepared from HA-EZH2 wildtype (WT), HA-EZH2 phospho-null (PN) S21A and HA-empty vector (EV) expressing SW480 cells treated with 250 μM H 2 O 2 for 30 minutes. Whole-cell extract (WCE) serves as a control for the chromatin extract. B . Western blots of chromatin lysate prepared from HA-EZH2 WT, HA-EZH2 S21D and HA-EV expressing SW480 cells. C . Western blots of chromatin lysate prepared from SW480 cells untreated and treated with 1 μM EZH2 inhibitor (EZH2i, GSK-503) for 72 hours or with 10 μM AKT inhibitor (AKTi, GSK-690693) for 48 hours and followed by no additional treatment or co-treatment with 250 μM H 2 O 2 for 30 minutes. D . Western blots of chromatin lysate prepared from FLAG-β-catenin WT and FLAG-β-catenin K49R expressing PTEN knockdown (KD) and EV expressing SW480 cells. WCE serves as a control for the chromatin extract.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: Western Blot, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Metagenomic heatmap for HA CUT&RUN prepared from HA-EZH2 wildtype (WT) and HA-EZH2 S21A expressing PTEN knockdown (KD) and empty vector (EV) SW480 cells. B . Metagenomic heatmap for FLAG ChIP-seq prepared from FLAG-β-catenin WT and FLAG-β-catenin K49R expressing PTEN KD and EV expressing SW480 cells. C. Average ChIP-seq read intensity for all peaks shown in B. D . Ridgeplot for differentially expressed genes (DEGs) in PTEN KD vs EV SW480 cells. E . Heatmap for DEGs in PTEN KD versus EV clustered manually based on the effect of EZH2 inhibitor (GSK503, 1 μM, 72 hours). F. Functional gene annotations for DEGs in cluster 1 and cluster 2 from E generated by Metascape. G . ChIP-seq gene tracks of representative DEGs in PTEN KD versus EV SW480 cells.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: Expressing, Plasmid Preparation, ChIP-sequencing, Functional Assay, Generated

Journal: bioRxiv

Article Title: Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer

doi: 10.1101/2023.01.31.526429

Figure Lengend Snippet: A . Barplot for the hallmark analysis for differentially expressed genes (DEGs) in cluster 2. B . Centplot for the hallmark analysis in B. C . FLAG-β-catenin ChIP-seq gene tracks of representative hallmark genes. D . Empty vector (EV) and PTEN knockdown (KD) cells were treated with DMSO or 2 μM EZH2 inhibitor (EZH2i, GSK-503) for 72 hours followed by plating cells in the upper chamber of a transwell insert. Brightfield images of crystal violet–stained migrated cells were taken after 48 hours. Scale bar = 200 microns. E . Quantification of migration normalized to migration counts for untreated EV cells. Results are represented as mean +/- SD. Significance was determined by one-way ANOVA with the Tukey multiple comparisons test. All significant comparisons are shown. ** P< 0.01. F . Model depicting the role of pS21-EZH2 in regulating AKT-mediated transcription through β-catenin methylation.

Article Snippet: For Western blot of endogenous proteins, anti-EZH2 [Cell Signaling Technology (CST, #5246, 1:1,000], anti–β-catenin (CST, #8480, 1:1,000), anti–TCF1 (CST, #2203, 1:1,000), anti-Rpb1 CTD (4H8) (CST, #2629, 1:1,000), anti-Vimentin (CST, #5741, 1:1,000), anti-Phospho-Akt (Ser473) (CST, #4060, 1:1,000), anti-total AKT (CST, #4691,1:1,000), anti-TCF1 (SC, sc-271453, 1:1,000), anti-H3K27me3 (CST,#9733, 1:1000), anti-H3(CST, #4499, 1:1000) anti-GAPDH (CST, #5174,1:1,000), anti-Phospho-Akt Substrate (RXXS*/T*) (CST, #9614, 1:1000), and anti-FLAG (sigma, F3165) antibodies were used.

Techniques: ChIP-sequencing, Plasmid Preparation, Staining, Migration, Methylation

Journal: Life Science Alliance

Article Title: Loss of dyskerin facilitates the acquisition of metastatic traits by altering the mevalonate pathway

doi: 10.26508/lsa.202201692

Figure Lengend Snippet: List of antibodies.

Article Snippet: EZH2 , 5246 , Cell Signaling , WB.

Techniques:

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) Chromosome localization and gene-coding sequence (CDS) of EZH2 in human. ( B ) The two domains (CXC domain and SET domain), five regions, and four compositional biases of EZH2. ( C ) The protein structure of EZH2 gene. ( D ) Conservation of EZH2 protein among various species. ( E ) The phylogenetic tree of EZH2 in different species. ( F ) The main location of EZH2 protein in cells. ( G ) The sites of EZH2 protein in the REH, U-2 OS, and SiHa cells.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Sequencing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) The expression levels of EZH2 in normal and tumor tissues in human body. ( B ) The mRNA expression levels of EZH2 in normal tissues (data from GTEx). ( C ) IHC-staining images from the HPA database displaying protein expression levels of EZH2 in the testis, esophagus, skin, and spleen tissues. ( D ) The mRNA expression levels of EZH2 in normal cell lines.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Expressing, Immunohistochemistry

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) EZH2 presenting moderate to strong nuclear staining in most cancer types. ( B ) IHC results of EZH2 protein assessed in lung cancer, colorectal cancer, head and neck cancer, as well as testis cancer. ( C ) The expression levels of EZH2 in different cancer cell lines.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Staining, Expressing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) The differences in mRNA expression levels of EZH2 in different normal and tumor tissues from TCGA+GTEx. ( B ). The expression differences of EZH2 protein in various normal and tumor tissues from CPTAC.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Expressing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) Summary of alterations in EZH2 expression in various cancer types. ( B ) The alteration frequency with mutation type. ( C ) Several mutated site information of EZH2 were exhibited by 3D structure. ( D ) The types and sites of EZH2 genetic mutations were noted in whole amino acid sequence.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Expressing, Mutagenesis, Sequencing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) Differential methylation of EZH2 promoter in ten cancer types. ( B ) Phosphorylation sites of EZH2 protein. ( C ) Differential phosphorylation of EZH2 protein at T487 site in GBM, LIHC, LUAD, as well as breast cancer.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Methylation

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A,B ) Heatmap showing correlations of EZH2 expression with infiltration by six immune cell types and three immunosuppressive cell types in various TCGA cancer types. ( C ) Scatter plots visualizing a no or inverse relationship between EZH2 expression and six tumor-infiltrating immune cells in GBM, LUSC, TGCT, and SARC. ( D ) Heatmap showing the roles of EZH2 expression in dysfunctional T-cell phenotypes of TCGA pan-cancer cohort.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Expressing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A,B ) Sensitivity and resistance to drugs targeting EZH2 on the basis of GDSC database (Red indicates drug resistant; blue indicates drug sensitive.). ( C ) Afuresertib IC50 values for EZH2 mutation. ( D,E ) The structural formula and 3D structure of Afuresertib. ( F ) Venetoclax IC50 values for EZH2 mutation. ( G ) The structural formula of Venetoclax.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Mutagenesis

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) PPI network for EZH2 and EZH2-binding proteins using GPS-Prot. ( B ) GO functional annotation analysis for EZH2-related genes. ( C ) KEGG pathway enrichment analysis for EZH2-related genes. ( D ) Coexpression network of EZH2 and its molecular partners via GeneMANIA. ( E ) The enriched gene sets in KEGG collection by the high EZH2 expression sample. ( F ) The enriched gene sets in KEGG by samples with low EZH2 expression. ( G ) Enriched gene sets in HALLMARK by samples of high EZH2 expression. ( H ) Enriched gene sets in HALLMARK by the low EZH2 expression. ( I ) The result of predicted miRNAs using two different databases based on Venn plots. ( J ) Complementary sequences of EZH2 and target miRNAs (i.e., hsa-let-7a-5p and hsa-let-7b-5p). ( K ) mRNA-miRNA-lncRNA network for EZH2.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Binding Assay, Functional Assay, Expressing

Journal: Bioscience Reports

Article Title: Multiomics characteristics and immunotherapeutic potential of EZH2 in pan-cancer

doi: 10.1042/BSR20222230

Figure Lengend Snippet: ( A ) STAD. ( B ) COAD. ( C ) LIHC. ( D ) PAAD. IHC further verifies the expression of EZH2 gene. ( E ) STAD. ( F ) COAD. ( G ) LIHC. ( H ) PAAD.

Article Snippet: These samples were incubated with 1:50 dilution of anti-EZH2 rabbit mAb (CST, #5246).

Techniques: Expressing

Journal: bioRxiv

Article Title: PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion

doi: 10.1101/2023.01.23.525196

Figure Lengend Snippet: Genetic and pharmacological disruption of polycomb proteins in BT-549 cells. (A) Expression levels for polycomb subunit-encoding genes (publicly available RNA-seq data from cBioPortal) is shown for TNBC-derived cell lines versus non-cancer mammary epithelial cells (HMEL). Mesenchymal: BT-549, MDA-MB-231, MDA-MB-436; basal: HCC2157, MDA-MB-468, HCC70, HCC1806; unclassified: BT-20, HCC1500. (B) Western blots of lysates from hTERT-HME1 (non-cancer) and BT-549 cells with antibodies against the indicated polycomb proteins or loading controls. (C) Overview of polycomb targeting strategies that were tested in BT-549 for comparison in this study. Genetic knock-down via siRNA against EZH2 and PCGF4 (BMI1) in BT-549 cells was confirmed by western blot. (D) PCA plot of normalized expression levels for 177 genes in all conditions (four independent treatments each) determined by a Nanostring assay. (E) Average baseline normalized expression of 177 genes (rows) for the control samples (DMSO and siCtrl) and log2 fold change expression values for all treatments. Genes symbols and numerical values are provided in Supplemental Table S1 . (F) H3K27me3 ChIP-seq data for BT-549 (DMSO-treated) from Lehmman et al. is shown in the transcription start site (TSS) plot, with rows corresponding to the order of rows in panel E.

Article Snippet: Primary staining was performed on separate blots with the following antibody dilutions in 5% BSA/TBST: rabbit anti-EZH2 1:1000 (Cell Signaling Technology, 5246S), rabbit anti-CBX2 1:500 (Bethyl Laboratories, A302-524A-T), rabbit anti-CBX4 1:1000 (Abcam, ab242149), rabbit anti- PCGF4/ BMI1 1:1000 (Cell Signaling Technology, 6964S), rabbit anti-VIN 1:1000 (Cell Signaling Technology, 13901S), rabbit anti-GAPDH 1:1000 (Cell Signaling Technology, 2118S).

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

Journal: bioRxiv

Article Title: PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion

doi: 10.1101/2023.01.23.525196

Figure Lengend Snippet: Transcription and epigenomic profiling of SRA-expressing BT-549 cells. (A) BT-549 cells were transfected with SRA-expressing plasmid DNA and analyzed with RNA-seq 24, 48, and 72 hours after transfection (2 replicates per condition). (B) Principal component analysis showed strong reproducibility of RNA-seq data from two biological replicates (independent transfections) per condition. (C) 122 SRA UpDEGs (rows) are sorted from low to high baseline expression. Log2 fold change at each time point post-transfection is shown in the color gradient map. The top 100 highly expressed genes are included for comparison. Transcription start site (TSS) plots for H3K27me3 ChIP-seq and ATAC-seq signals show mean signals across 10 Kb regions centered at the TSS’s of each group of genes. Expression values for SRA UpDEGs and the top 100 highly expressed genes are provided in Supplemental Table S2 . (D) The TSS plot shows mean H3K27me3 ChIP-seq signals for BT-549 cells control-treated with DMSO or treated with the EZH2 inhibitor Tazemetostat. (E) Wiggle (WIG) tracks show H3K27me3 ChIP-seq data from DMSO (black) or Tazemetostat-treated (gray) BT-549 cells. H3K27me3 ChIP -seq peaks and annotations for chromatin features from the ENCODE Broad ChromHMM dataset are shown for six representative SRA UpDEGs that are located near strong enhancers.

Article Snippet: Primary staining was performed on separate blots with the following antibody dilutions in 5% BSA/TBST: rabbit anti-EZH2 1:1000 (Cell Signaling Technology, 5246S), rabbit anti-CBX2 1:500 (Bethyl Laboratories, A302-524A-T), rabbit anti-CBX4 1:1000 (Abcam, ab242149), rabbit anti- PCGF4/ BMI1 1:1000 (Cell Signaling Technology, 6964S), rabbit anti-VIN 1:1000 (Cell Signaling Technology, 13901S), rabbit anti-GAPDH 1:1000 (Cell Signaling Technology, 2118S).

Techniques: Expressing, Transfection, Plasmid Preparation, RNA Sequencing Assay, ChIP-sequencing