Journal: Oncology Letters

Article Title: miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2

doi: 10.3892/ol.2013.1173

Figure Lengend Snippet: EZH2 was inversely correlated with miR-26a levels. (A) The expression levels of miR-26a and EZH2 in 5-8F cells transfected with LV-control and LV-miR-26a. ** P<0.01 compared with the control group. (B) The expression of EZH2 protein in cells transfected with LV-miR-26a was decreased compared with the control. (C) Immunohistochemistal staining of EZH2 in primary liver tumor tissues of NPC metastasis-bearing mice. The representative images are presented (magnification, ×100). EZH2, enhancer of zeste homolog 2; NPC, nasopharyngeal carcinoma.

Article Snippet: The membrane was incubated with a rabbit monoclonal antibody against human EZH2 (1:500 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) followed by HRP-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and detected by chemiluminescence.

Techniques: Expressing, Transfection, Control, Staining

Journal: Oncology Letters

Article Title: miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2

doi: 10.3892/ol.2013.1173

Figure Lengend Snippet: Immunohistochemical detection of EZH2 in primary tumors in the control and miR-26a groups.

Article Snippet: The membrane was incubated with a rabbit monoclonal antibody against human EZH2 (1:500 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) followed by HRP-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and detected by chemiluminescence.

Techniques: Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation

Journal: Clinical Epigenetics

Article Title: H3K27me3 and EZH2 expression in melanoma: relevance for melanoma progression and response to immune checkpoint blockade

doi: 10.1186/s13148-020-0818-7

Figure Lengend Snippet: H3K27me3 and EZH2 IHC expression in melanoma metastases prior to anti-PD-1 inhibition

Article Snippet: The primary antibodies used were a rabbit monoclonal anti-H3K27me3 antibody (dilution 1:200, Cell Signaling Technology, C36B11) and a rabbit monoclonal anti-EZH2 antibody (dilution 1:50, Cell Signaling Technology, D2C9).

Techniques: Expressing, Inhibition

Journal: Clinical Epigenetics

Article Title: H3K27me3 and EZH2 expression in melanoma: relevance for melanoma progression and response to immune checkpoint blockade

doi: 10.1186/s13148-020-0818-7

Figure Lengend Snippet: H3K27me3 and EZH2 expression of melanoma metastases prior to anti-PD-1 therapy. a H3K27me3 and EZH2 expression of melanoma metastases (in %) in correlation to response to anti-PD-1 inhibition. Significance was determined by Χ 2 test for categorical data. b Percentage of H3K27me3-positive melanoma cells in correlation to percentage of EZH2-positive melanoma cells. Significance was determined by Spearman’s rank correlation test ( p = .002)

Article Snippet: The primary antibodies used were a rabbit monoclonal anti-H3K27me3 antibody (dilution 1:200, Cell Signaling Technology, C36B11) and a rabbit monoclonal anti-EZH2 antibody (dilution 1:50, Cell Signaling Technology, D2C9).

Techniques: Expressing, Inhibition

Journal: Cell

Article Title: The bone microenvironment invigorates metastatic seeds for further dissemination

doi: 10.1016/j.cell.2021.03.011

Figure Lengend Snippet: (A) Levels of EZH2 signature genes (GSVA) in bone entrained and other SCP21 cells. (B) Levels of EZH2 signature genes in bone entrained-SCP21 cells after different passages in vitro. (C) Percentage of ALDH1+ population in bone entrained-SCP21 cells at different passages. (D) Representative western blotting of proteins in bone entrained-SCP21 cells after different passages. (E-G) The schematic diagram and representative BLI images (E), normalized BLI intensity at day 7 (F), and the colonization kinetics (G) of BoM-SCP21 cells with in vitro EPZ011989 (EPZ) treatment before IC injection. Non-treated BoM-SCP21 cells were used as control. N (# of mice) = 15 (-EPZ); 9 (+EPZ). (H) Comparison of ALDH1+ cells in EPZ treated and non-treated BoM-MCF7-SCP2 cells by flow cytometry. N (# of replicate) =3. (I-K) Representative BLI images (I), normalized BLI intensity at day 7 (J), and the colonization kinetics (K) of BoM-MCF7-SCP2 cells with in vitro treatment of EPZ before IC injection. Non-treated BoM-MCF7-SCP2 cells were used as control. N (# of mice) = 10 (-EPZ); 7 (+EPZ). (L) Experimental design assessing the multi-site metastases from bone lesions with inducible depletion of EZH2. (M) Growth kinetics of the primary bone lesions in mice receiving doxycycline or control water, assessed by in vivo BLI imaging. BLI intensities at right hindlimbs were normalized to the mean intensity at day 0. N (# of mice) = 10 for each arm. (N) Heat map of ex vivo BLI intensity and status of metastatic involvement in tissues from animals with EZH2 depleted or control bone metastases. Data are represented as mean ± SEM in F, G, J, K, and M. P values were assessed by student t-test in A, F, and J; by test for linear trend following repeat measure one-way ANOVA in B and C; by LSD test following two-way ANOVA in G, K and M; by ratio paired t-test in H; by Fisher’s exact test on the ratio of metastatic involvement and Mann-Whitney test on BLI intensity in N. See also Figure S7.

Article Snippet: Ezh2 (D2C9) XP® Rabbit mAb, 1:1000 in western blotting, 1:100 in immunofluorescent staining , Cell Signaling Technology , Cat# 5246S RRID: AB_10694683.

Techniques: In Vitro, Western Blot, Injection, Control, Comparison, Flow Cytometry, In Vivo, Imaging, Ex Vivo, MANN-WHITNEY

Journal: Cell

Article Title: The bone microenvironment invigorates metastatic seeds for further dissemination

doi: 10.1016/j.cell.2021.03.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Ezh2 (D2C9) XP® Rabbit mAb, 1:1000 in western blotting, 1:100 in immunofluorescent staining , Cell Signaling Technology , Cat# 5246S RRID: AB_10694683.

Techniques: In Vivo, Western Blot, Staining, Recombinant, Multiplex Assay, Sequencing, RNA Sequencing, Derivative Assay, Cloning, shRNA, Software

Journal: Oncology Letters

Article Title: Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma

doi: 10.3892/ol.2020.12212

Figure Lengend Snippet: Representative images of staining for KDM6A, EZH2 and H3K27me3 in LG and HG urothelial carcinoma. Magnification ×100. KDM6A, lysine demethylase 6A; EZH2, histone-lysine N-methyltransferase EZH2; LG, low grade; HG, high grade.

Article Snippet: The first slide was stained with hematoxylin (5 min) and eosin (1–3 min) at room temperature for hematoxylin to confirm the presence of tumor cells, and subsequent slides were used to evaluate the reactivity of primary antibodies, including rabbit polyclonal anti-KDM6A antibody (1:100; cat. no. ab36938; Abcam), mouse monoclonal anti-EZH2 antibody (1:150; cat. no. 6A10; Origene Technologies, Inc.) and rabbit polyclonal anti-H3K27me3 antibody (1:150; cat. no. A2363; ABclonal Biotech Co., Ltd.).

Techniques: Staining

Journal: Oncology Letters

Article Title: Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma

doi: 10.3892/ol.2020.12212

Figure Lengend Snippet: Clinicopathological characteristics associated with KDM6A, EZH2 and H3K27me3 expression.

Article Snippet: The first slide was stained with hematoxylin (5 min) and eosin (1–3 min) at room temperature for hematoxylin to confirm the presence of tumor cells, and subsequent slides were used to evaluate the reactivity of primary antibodies, including rabbit polyclonal anti-KDM6A antibody (1:100; cat. no. ab36938; Abcam), mouse monoclonal anti-EZH2 antibody (1:150; cat. no. 6A10; Origene Technologies, Inc.) and rabbit polyclonal anti-H3K27me3 antibody (1:150; cat. no. A2363; ABclonal Biotech Co., Ltd.).

Techniques: Expressing, Significance Assay

Journal: Oncology Letters

Article Title: Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma

doi: 10.3892/ol.2020.12212

Figure Lengend Snippet: Univariate and multivariate analyses of CSS and DFS in patients with upper tract urothelial carcinoma (n=108).

Article Snippet: The first slide was stained with hematoxylin (5 min) and eosin (1–3 min) at room temperature for hematoxylin to confirm the presence of tumor cells, and subsequent slides were used to evaluate the reactivity of primary antibodies, including rabbit polyclonal anti-KDM6A antibody (1:100; cat. no. ab36938; Abcam), mouse monoclonal anti-EZH2 antibody (1:150; cat. no. 6A10; Origene Technologies, Inc.) and rabbit polyclonal anti-H3K27me3 antibody (1:150; cat. no. A2363; ABclonal Biotech Co., Ltd.).

Techniques:

Journal: Oncology Letters

Article Title: Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma

doi: 10.3892/ol.2020.12212

Figure Lengend Snippet: Pairwise association among the KDM6A, EZH2 and H3K27me3 expression levels in upper tract urothelial carcinoma.

Article Snippet: The first slide was stained with hematoxylin (5 min) and eosin (1–3 min) at room temperature for hematoxylin to confirm the presence of tumor cells, and subsequent slides were used to evaluate the reactivity of primary antibodies, including rabbit polyclonal anti-KDM6A antibody (1:100; cat. no. ab36938; Abcam), mouse monoclonal anti-EZH2 antibody (1:150; cat. no. 6A10; Origene Technologies, Inc.) and rabbit polyclonal anti-H3K27me3 antibody (1:150; cat. no. A2363; ABclonal Biotech Co., Ltd.).

Techniques: Expressing

Journal: Endocrine-Related Cancer

Article Title: The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors

doi: 10.1530/erc-13-0497

Figure Lengend Snippet: Figure 1 EZH2 mRNA and protein expression in hyperparathyroid tumors. (A) Real- time quantitative RT-PCR analysis of EZH2 in normal parathyroid tissues and parathyroid tumors. pHPT denotes parathyroid adenomas and sHPT secondary hyperplastic parathyroid glands. The relative expression level of the one normal parathyroid tissue that was obtained from glands inadvertently removed in conjunction with thyroid surgery was arbitrary set to 1.0. The four normal parathyroid gland biopsies from HPT patients displayed relative EZH2 expression levels of 0.8, 2.1, 2.2, and 3.1. (B) Western blotting analysis of EZH2. Tumors with relatively high mRNA levels (upper panel) or with mRNA levels in the normal range (lower panel). Samples no. 5, 7, 8, 14, and 15 displayed EZH2 gene amplification. (C) Immunohistochem- ical analysis of EZH2. Paraffin-embedded sections were stained using a rabbit MAB (two upper panels) or without primary antibody (lower panel right). Western blotting of the same specimens is also shown.

Article Snippet: Paraffin-embedded specimens were stained as described (Björklund et al. 2007b) using an anti-EZH2 rabbit MAB (Cell Signaling Technology, Inc., EZH2 (D2C9), catalog no. 5246).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Endocrine-Related Cancer

Article Title: The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors

doi: 10.1530/erc-13-0497

Figure Lengend Snippet: Figure 4 Wnt/b-catenin signaling on EZH2 knockdown. (A) Transient transfection of siControl and siEZH2 to the sHPT-1 parathyroid tumor cell line and determination of AXIN2 (left panel) and CYCLIN D1 (right panel) expression by real-time quantitative RT-PCR analysis. (B) Western blotting analysis of transiently siRNA-transfected sHPT-1 cells using the anti-active b-catenin antibody. This antibody is specific for transcriptionally active (non- phosphorylated) b-catenin (van Noort et al. 2002). (C) Transient transfec- tion of siControl, siLRP5, and siEZH2 and determination of LRP5 and EZH2 relative mRNA expression by real-time quantitative RT-PCR analysis.

Article Snippet: Paraffin-embedded specimens were stained as described (Björklund et al. 2007b) using an anti-EZH2 rabbit MAB (Cell Signaling Technology, Inc., EZH2 (D2C9), catalog no. 5246).

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Endocrine-Related Cancer

Article Title: The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors

doi: 10.1530/erc-13-0497

Figure Lengend Snippet: Figure 5 Summary of the results and possible mechanisms. EZH2 protein over- expression caused by EZH2 gene amplification and increased mRNA level or by indirect mechanisms such as EZH2 protein stability and by direct or indirect regulation of EZH2 gene expression by the c-Myc oncoprotein (Bjo¨ rklund et al. 2007b, Koh et al. 2011). Deregulated increased EZH2 protein level could result in activation of the Wnt/b-catenin signaling pathway by repression of AXIN2 (Cheng et al. 2011) and accumulation of transcriptionally active (nonphosphorylated) b-catenin, with increased expression of the b-catenin target gene CYCLIN D1 (Bjo¨ rklund et al. 2007a) and proliferation. Repression of the HIC1 tumor suppressor gene in parathyroid tumors involved EZH2 and H3K27 methylation (Svedlund et al. 2012).

Article Snippet: Paraffin-embedded specimens were stained as described (Björklund et al. 2007b) using an anti-EZH2 rabbit MAB (Cell Signaling Technology, Inc., EZH2 (D2C9), catalog no. 5246).

Techniques: Over Expression, Gene Expression, Activation Assay, Expressing, Methylation

Journal: International journal of molecular sciences

Article Title: USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

doi: 10.3390/ijms241813780

Figure Lengend Snippet: Figure 5. Impact of USP7 inhibition on expression and ubiquitination of EZH2. (A–E) Western blot analysis of USP7, EZH2, GAPDH, and actin protein expression levels in SK-N-SH (A), NB-10 (B), LAN-5 (C), NBL-S (D), and IMR-32 (E) NB cells after treatment with DMSO control or after increasing doses of Almac4. Band densitometry was performed using ImageJ and band densities for USP7 and EZH2 were normalized to control protein band densities (GAPDH or actin) and plotted (F) IMR-32 NB cells were treated with DMSO or increasing concentrations of Almac4, and immunoprecipitated EZH2 was analyzed by Western blot for lysine-48-linked (K48) ubiquitin. (G) Western blot analysis of USP7, EZH2, and GAPDH protein expression levels in SK-N-SH, NB-10, CHP-212, LAN-5, IMR-32, and NBL-S NB cells after treatment with DMSO or after 1 µM of Almac4 for 48 h.

Article Snippet: Rabbit monoclonal antibodies anti-EZH2 (5246S), anti-HAUSP (4833S), anti-p53 (2527S), anti-GAPDH (5174S), anti-vinculin (13901S), and rabbit polyclonal antibodies anti-PARP (9542S), anti-caspase-3 (9262S), anti-cleaved caspase-3 (9661S), anti-ubiquitin (3933S), anti-K48-linkage polyubiquitin (8081S), and mouse monoclonal antibody anti-p53 (18032S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Expressing, Ubiquitin Proteomics, Western Blot, Control, Immunoprecipitation

Journal: International journal of molecular sciences

Article Title: USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

doi: 10.3390/ijms241813780

Figure Lengend Snippet: Figure 6. Impact of USP7 depletion on expression of MDM2, p53, and EZH2. (A,B) (Upper panels) Parental SK-N-SH (A) and NB-10 (B) NB cells and matching cell lines with USP7 depletion using 3 different shRNAs in each cell line were analyzed by live cell imaging for cell confluence over time. Relative cell confluence over 72 h is shown. (Lower panels) Western blot analyses of USP7 protein expression levels in matching NB cell lines from above. Band densitometry was performed using ImageJ and band densities for USP7 were normalized to control protein band densities (GAPDH) and plotted. (C,D) Western blot analysis of USP7, MDM2, p53, EZH2, and GAPDH protein expression levels in SK-N-SH (C) and NB-10 (D) parental and USP7 knockdown cell lines. Band densitometry was performed using ImageJ and band densities for USP7, MDM2, p53, and EZH2 were normalized to control protein band densities (GAPDH) and plotted.

Article Snippet: Rabbit monoclonal antibodies anti-EZH2 (5246S), anti-HAUSP (4833S), anti-p53 (2527S), anti-GAPDH (5174S), anti-vinculin (13901S), and rabbit polyclonal antibodies anti-PARP (9542S), anti-caspase-3 (9262S), anti-cleaved caspase-3 (9661S), anti-ubiquitin (3933S), anti-K48-linkage polyubiquitin (8081S), and mouse monoclonal antibody anti-p53 (18032S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Live Cell Imaging, Western Blot, Control, Knockdown

Journal: International journal of molecular sciences

Article Title: USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

doi: 10.3390/ijms241813780

Figure Lengend Snippet: Figure 7. Impact of USP7 inhibition and depletion on expression and ubiquitination of N-myc. (A,B) Western blot analysis of USP7, EZH2 N-Myc, and GAPDH protein expression levels in SK-N- SH (A) and NB-10 (B) parental and USP7 knockdown NB cell lines. (C) IMR-32 NB cells were treated with DMSO or increasing concentrations of Almac4, and immunoprecipitated N-myc was analyzed by Western blot for lysine-48-linked (K48) ubiquitin, FBW7, and EZH2. (D) Cell lysates of IMR-32 NB cells treated with DMSO or increasing concentrations of Almac4 were analyzed by Western blot for USP7, EZH2, FBW7, N-Myc, and actin protein expression levels.

Article Snippet: Rabbit monoclonal antibodies anti-EZH2 (5246S), anti-HAUSP (4833S), anti-p53 (2527S), anti-GAPDH (5174S), anti-vinculin (13901S), and rabbit polyclonal antibodies anti-PARP (9542S), anti-caspase-3 (9262S), anti-cleaved caspase-3 (9661S), anti-ubiquitin (3933S), anti-K48-linkage polyubiquitin (8081S), and mouse monoclonal antibody anti-p53 (18032S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Expressing, Ubiquitin Proteomics, Western Blot, Knockdown, Immunoprecipitation