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: 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: The Journal of Biological Chemistry

Article Title: Polycomb Repressive Complex 2 (PRC2) Protein ESC Regulates Insect Developmental Timing by Mediating H3K27me3 and Activating Prothoracicotropic Hormone Gene Expression *

doi: 10.1074/jbc.M113.482497

Figure Lengend Snippet: Expression and cellular localization of ESC, interaction between ESC and E(z), and methylation levels of histone H3 in brains of nondiapause- and diapause-destined pupae. A, shown is a Western blot analysis of Har-ESC transiently expressed in Sf9 cells. This is a schematic diagram of full-length ESC (ESC-FL), N-terminal ESC (ESC-N), and C-terminal ESC (ESC-C), which were used for transfection, respectively. NLS, nuclear location site, is in red; WD domain is in blue; full-length ESC-GFP at 1, 2, and 5 μg, N-terminal-GFP at 0.5 and 1 μg, and C-terminal-GFP at 1 and 2 μg transiently expressed in Sf9 cells. B, cellular localization of transiently expressed recombinant ESC. Hoechst 33342 staining shows the cell nuclei. Full-length ESC-GFP, N-terminal ESC-GFP, and C-terminal ESC-GFP were transfected into Sf9 cells; pIZ-V5-GFP (EGFP) was used as a control. Scale bar, 20 μm. C, ESC is physically associated with E(z). The HzAm1 cell extracts were immunoprecipitated (IP) with anti-ESC antibody, and the immunoblot (IB) was performed with anti-EZH2 antibody. Rabbit serum (IgG) served as a negative control. The 84-kDa band is E(z), and the 52-kDa band is IgG heavy chain. D, a Western blot analysis of H3K27me3, H3K4me3, and H3K27Ac detects methylation or acetylation of histone H3. The antibody against total H3 served as the loading control.

Article Snippet: Antibody against full-length human EZH2 (SAB1405776, Sigma) was used against E(z) protein; H3 (ab1791), H3K27Ac (ab4729), H3K4me3 (ab8580) were purchased from Abcam, and H3K27me3 (17-622) was from Millipore.

Techniques: Expressing, Methylation, Western Blot, Transfection, Recombinant, Staining, Immunoprecipitation, Negative Control

Journal: Human Mutation

Article Title: Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

doi: 10.1002/humu.22946

Figure Lengend Snippet: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro . Histone methyltransferase reactions were performed using 2 μg purified core histones and 0.67 μM 3 H‐S‐adenosyl‐methionine ( 3 H‐SAM). Each reaction was incubated with 250 ng of either wild‐type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3 H‐labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for each complex: EZH2 WT • , p.(Phe672Ile) × , p.(Pro132Ser) ★ , p.(Tyr153del) △, p.(His694Tyr) ▽, p.(Glu745Lys) ▴, p.(Ala682Thr) ▾, p.(Arg684Cys) ▪, p.(Tyr133Cys) □, and p.(Asp185His) ◇. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t‐test showed statistically significant difference between the two groups (P value < 0.0001). B : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One‐way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Article Snippet: To test our hypothesis, we designed recombinant human EZH2 proteins, had them preassembled into PRC2 complexes (BPS Bioscience, San Diego CA), and tested their activity in vitro using a well‐accepted in vitro assay [Ernst et al., ; Yap et al., ; Score et al., ].

Techniques: Activity Assay, In Vitro, Purification, Incubation, Mutagenesis, Labeling, Standard Deviation, Positive Control, Negative Control

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: The expression vector PIRVNeoSV containing the human c-Myc cDNA coding sequence (c-Myc) was kindly provided by Dr. Giuseppe Giannini (Sapienza University, Rome, Italy). pCMV3-HA vector containing the human EZH2 coding sequence (HA-EZH2) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL enhances PRC2 activity in vitro. A, Coomassie Blue staining of PRC2 complexes (containing EZH2, SUZ12, and EED) purified from Sf9 cells. B, MNase digestion of reconstituted oligonucleosomes resolved by 2% agarose gel. Left panel: lane 1 shows the pure pG5E4 plasmid DNA, and lane 2 shows band shift of pG5E4 DNA assembly into oligonucleosomes. Right panel: equimolar amounts of reconstituted oligonucleosomes were digested with increasing amounts of MNase (Sigma). The DNA was isolated and subjected to electrophoresis on a 2% agarose gel in the presence of ethidium bromide. Partial MNase digestion (oligonucleosome: MNase = 2 μg: 0.5 μl) generated a nucleosomal DNA ladder with visible mono-, di-, and trinucleosomal fragments, which are indicated by corresponding numbers of asterisks. Mononucleosomal DNA runs as a 147 bp fragment. C, CDYL stimulates PRC2 activity in vitro. Reconstituted recombinant oligonucleosomes were incubated with EZH2/SUZ12/EED complexes (PRC2-core) in the absence or presence of increasing amounts of baculovirus generated CDYL and histone methyltransferase activity was determined by standard HMT assays. The reaction products were examined by Western blotting with the antibodies indicated on the right. Ponceau staining of histones is shown in the bottom panel to show equal amounts of substrates used in each reaction. D, CDYL only stimulates PRC2 methyltransferase activity toward oligonucleosome, but not mononucleosome substrates. Reconstituted Xenopus oligonucleosomes were digested with MNase (oligonucleosome: MNase = 1 μg: 1 μl) at room temperature for 5 min. This treatment yielded mainly mononucleosomes (see Fig. 4B). Equal amounts of mononucleosomes were used as substrates for the HMT assay in the top panel, whereas equal amounts of undigested oligonucleosomes were used as substrates in the bottom panel. Commercially available EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (PRC2-full) were used to provide methyltransferase activity as indicated. The mild increase of PRC2 activity seen upon CDYL addition in the top panel was mainly due to incomplete digestion of oligonucleosomes (see Fig. 4B). E, binding affinity between CDYL and H3K27me3 is much stronger than the affinity between EED and H3K27me3. In the top panel, histone peptide binding assays show that CDYL, but not EED, binds to H3K27me2 when the same amounts of FLAG-tagged proteins (0.5 μg) were used in the assay. To compare the binding affinity for H3K27me3, 0.2, 0.4, or 1 μg of baculovirus-expressed FLAG-CDYL and 0.6, 1.2, or 3 μg of FLAG-EED proteins were used in the peptide binding assay. Ten percent of total proteins were used as loading controls (middle panel). Peptide-protein complexes were pulled down by streptavidin beads and bound proteins were examined by Western blotting using anti-FLAG antibodies (bottom panel). Ponceau staining of baculovirus-expressed FLAG-CDYL and FLAG-EED is shown in the right panel.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Activity Assay, In Vitro, Staining, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Isolation, Electrophoresis, Generated, Recombinant, Incubation, Western Blot, HMT Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL is physically associated with the PRC2 complex. A, in vivo immunoprecipitation. Top two panels: endogenous IP of MCF-7 cell lysates using antibodies against CDYL. Antibodies (EZH2 or SUZ12) used for Western blotting are indicated on the right. Bottom panel: MCF-7 cells were transfected with a FLAG-CDYL construct and subjected to co-IP assays after 48 h. Cell protein extracts were immunoprecipitated with polyclonal antibodies against EED, and blotted with monoclonal anti-FLAG antibodies. An immunoglobulin G (IgG) control is included in each experiment. B, GST pull-down assays. Purified GST or GST-CDYL proteins immobilized on glutathione Sepharose 4B beads were incubated with in vitro translated EZH2, SUZ12, or EED. Bound proteins were detected with monoclonal anti-EZH2 antibodies (top panel) or anti-MYC tag antibodies (lower two panels). C, Superose 6 gel filtration analysis of the MCF-7 nuclear extracts. Migration of molecular markers is indicated above the panels and the antibodies for Western blotting are indicated on the right. Equal volumes from each fraction were analyzed. D, similar FPLC experiments as in C using a Superdex 200 10/300 GL column. The chromatographic fractions were analyzed by Western blotting using the indicated antibodies. Bottom panel: HMT assays were performed using the indicated eluted fractions. Recombinant Xenopus H3 proteins were used as substrates, and the reaction products were analyzed by Western blotting with anti-H3K27me3 antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Transfection, Construct, Co-Immunoprecipitation Assay, Purification, Incubation, In Vitro, Filtration, Migration, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Mapping the domains responsible for the interaction between CDYL and EZH2. A, schematic drawing of CDYL protein. CDYL deletion mutants including del1 (1–309 aa), del2 (1–60 aa, the chromodomain), del3 (61–545 aa), del4 (310–545 aa, the coAP domain), and del5 (61–309 aa) were fused to GST. B, GST pull-down experiments were performed with in vitro translated FLAG-EZH2 and purified GST or GST-CDYL deletion mutants. The precipitated complexes were examined by Western blotting using monoclonal anti-EZH2 antibodies (the upper panel). Only CDYL mutants containing the middle region from 61–309 aa (del1, del3, del5) efficiently pulled down EZH2. The lower panel shows the Ponceau staining of purified GST fusion proteins added to the reaction. The arrows indicate the positions of the respective GST fusion proteins as labeled on the top. C, schematic drawing of EZH2 protein. EZH2 deletion mutants (del1 to del5) were cloned into the pGBKT7 plasmid, which contains a c-Myc epitope tag and can be transcribed/translated in vitro. Del8 and Del9 were fused to GST. D, GST pull-down experiments were performed with in vitro translated Myc-EZH2 deletion mutants and purified GST or GST-CDYL in the left panels. Right panel: GST pull-down assays were performed with in vitro translated Myc-CDYL, which was incubated with purified GST, GST-del8, GST-del9, or GST-SET8 (negative control protein). Bound proteins were examined by Western blotting using monoclonal anti-Myc antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vitro, Purification, Western Blot, Staining, Labeling, Clone Assay, Plasmid Preparation, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Validation of common target genes of CDYL and PRC2. A, quantitative ChIP assays were performed in MCF-7 cells with primer pairs specific to indicated gene promoters (see supplemental Table S1). Normal rabbit IgG, as well as polyclonal antibodies against CDYL, EZH2, and H3K27me3 were used to immunoprecipitate the protein-DNA complex. B, conventional semi-quantitative ChIP assays performed at the MYT1 and BASE promoters. C, CDYL and PRC2 exist in the same protein complex at the MYT1 and BASE promoters. ChIP and re-ChIP experiments were performed with the indicated antibodies and primer pairs. D, CDYL expression was efficiently knocked down by specific siRNAs. Non-silencing or CDYL specific siRNAs were transfected into MCF-7 cells. Total proteins were extracted and the expression of CDYL and EZH2 proteins were examined by Western blotting. Actin protein levels were measured to indicate equal loading of protein lysates. E, CDYL is required for PRC2 chromatin targeting at the MYT1 and BASE promoters. MCF-7 cells were transfected with control siRNA or CDYL-specific siRNA. 48 hours after the transfection, cell lysates were collected, and ChIP experiments were performed using the indicated antibodies. Real-time PCR assays were performed for the measurement. F, CDYL mainly represses the expression of target genes. MCF-7 cells were transfected with control or CDYL-specific siRNAs. Total RNAs were prepared and the mRNA levels of the indicated genes were examined by real-time RT-PCR. The data were normalized against the expression of GAPDH. Each bar represents the mean ± S.D. for triplicate measurements.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 2. RACGAP1 overexpression promoted neuroendocrine transformation in prostate cancer. (A) Western blot analysis showed that RACGAP1 was upregulated in NEPC-like cells. (B) Immunohistochemical study of different types of prostate cancer tissues showed that RACGAP1 was highly expressed in NEPC. (C) Data for quantified immunohistochemistry in adjacent normal tissues (n=10), tumor tissues (n=10) and NEPC (n=7) of prostate cancer are shown as mean + SD. (D) Enzalutamide (MDV3100) induced the production of RACGAP1, and DHT partially reversed this effect. (E) Western blot analysis showed the protein expression of RACGAP1, CHGA, and SYP in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days. (F) The mRNA level of RACGAP1, NCAM, CHGA, SYP, and NSE in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days were determined by qRT-PCR analysis. (G) RACGAP1 and NE markers (CHGA, NCAM, NSE, and SYP) in C4-2 cells following transient transfection with control (shNC) or RACGAP1 shRNA (sh1, sh2), as detected by qRT-PCR. (H) RACGAP1 and NE markers (CHGA, SYP) in C4- 2B cells following transient transfection with RACGAP1 or an empty vector, as detected by qRT-PCR. (I) Protein

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Over Expression, Transformation Assay, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Plasmid Preparation

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 5. RACGAP1 protein interacts with the EZH2 protein and increases the protein stability of EZH2. (A) Analysis of protein-protein interaction (PPI) information shows that RACGAP1 may directly interact with EZH2. (B) GSEA shows that high RACGAP1 expression was enriched in the EZH2-TARGET pathway. (C) RNA levels of RACGAP1 and EZH2 in NE-like cells treated with RACGAP1 or empty vector (CON), as determined by qRT-PCR. (D) Western blot was performed to show protein expression of RACGAP1 and E2F1 in NE-like cells following control, RACGAP1, RACGAP1 shRNA1 (sh1), or RACGAP1 shRNA1 (sh2) transfection. (E) Total cell lysates of NE-like cells were immunoprecipitated with anti-EZH2 or anti- RACGAP1 antibodies and blotted with corresponding antibodies. (F) Representative immunofluorescence images of RACGAP1 and EZH2 protein localization in PC3 cells. (G) C4-2B-N cells and PC3 cells were transiently transfected with CON or RACGAP1 and supplied with 10 mmol/L cycloheximide (CHX), and then, total cell lysates were collected at 0, 8, 16, and 24 h after treatment. Western blot analysis was used to measure protein levels. (H) Protein expression analysis was used to calculate the half-life of EZH2 protein for C4-2B-N and PC3 cells. (I) Cells transiently transfected with RACGAP1 knockdown were treated with vehicle (DMSO), chloroquine (50 μm), or MG132 (20 μm) for 12 h. Western blotting was used to detect the protein level of RACGAP1. Bar graphs show the statistical analysis of three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Transfection, Immunoprecipitation, Immunofluorescence, Knockdown

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 6. RACGAP1 stabilizes EZH2 protein expression in the ubiquitin-proteasome pathway and affects NED in prostate cancer by regulating EZH2. (A) PC3 and DU145 cells were treated with FLAG-RACGAP1 for 48 h. Total cell lysates were subjected to immunoprecipitation with EZH2 antibody and blotted with an anti- ubiquitin antibody. (B) 293T cells were co-transfected with GFP-EZH2, Myc-ubi, and different doses of FLAG- RACGAP1 (0, 2, 4 µg) for 48 h. Total cell lysates were subjected to immunoprecipitation with a GFP antibody and blotted with an anti-Myc antibody. (C) PC3 cells were transfected for rescue experiments, using PC3 cells with an shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid, and cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid. Western blotting was used to detect proteins using the indicated antibodies. We transfected PC3 and DU145 cell lines for rescue experiments, using cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus (RA) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2, and cells with the RACGAP1 expression lentivirus (RA) and shEZH2. (D) Western blotting was used to detect proteins using the indicated antibodies. (E) Transwell assay for indicated PC3 cells (magnification: 100×). Bar graphs showing the statistical analysis of three independent experiments. ***, p < 0.001, **, p < 0.01, *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Western Blot, Transwell Assay

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Primers for reverse transcription-quantitative polymerase chain reaction analysis.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Protein expression levels of EZH2 and ROCK1 from 5–8F cells determined using western blot analysis. 5–8F cells were treated with COE (0, 12.5, 25 and 50 µg/ml) or 2 µM DZNeP. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05. EZH2, enhancer of zeste homolog 2; ROCK1, Rho-associated coiled coil-containing protein kinase 1; COE, Celastrus orbiculatus extract; DZNeP, 3-Deazaneplanocin A.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction determination of relative EZH2 and ROCK1 mRNA expression in 5–8F cells. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05. (A) Relative EZH2 mRNA expression. (B) Relative ROCK1 mRNA expression. EZH2, enhancer of zeste homolog 2; ROCK1, Rho-associated coiled coil-containing protein kinase 1; DZNeP, 3-Deazaneplanocin A; COE, Celastrus orbiculatus extract.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation