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: 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: 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: 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: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: AD-MSC-derived exosomes promote the autophagy and proliferation of trophoblasts in hypoxia condition. (A) Representative CCK-8 assay results for JEG-3 and HTR-8 cells are shown. Trophoblast cells were treated with AD-MSC exosomes under hypoxic conditions. (B) Representative EdU assay results for trophoblasts are shown. The histogram of EdU positive trophoblasts that treated with AD-MSC exosomes under hypoxic conditions was shown. (C) Whole cell lysates from trophoblast cells were subjected to western blotting to analyze and quantificate LC3, BECN1 and p62 levels. β-actin was included as a loading control. (D) Immunofluorescence analysis showed autophagosomes in the cytoplasm of trophoblast cells treated with AD-MSC exosomes under hypoxic conditions (scale bar, 25 μm). (E) Significant decreases in EZH2, mTOR and S6K1 mRNA levels were found in JEG-3 and HTR-8 cells treated with AD-MSC exosomes by qRT-PCR. (F) Whole cell lysates from trophoblast cells were subjected to western blotting to analyze EZH2, mTOR, S6K1, p-mTOR and p-S6K1 levels and quantificate EZH2 expression. β-actin was included as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Derivative Assay, CCK-8 Assay, EdU Assay, Western Blot, Control, Immunofluorescence, Quantitative RT-PCR, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: The inhibition of EZH2 induced the increase of trophoblast autophagy and inhibition of mTOR pathway under hypoxic conditions. (A–C) The protein expression level of LC3, BECN1 and P62 levels were examined in JEG-3 and HTR-8 cells treated with GSK126, AD-MSC exosomes or Baf A1 for 24 h by western blot. The protein expression levels were quantified by densitometry. (D) Cell proliferation was evaluated by a CCK-8 assay. The trophoblast cell lines were treated with GSK126 or Baf A1 and with or without AD-MSC exosomes under hypoxic conditions. (E) The EdU positive percent of trophoblast cell lines were tested under hypoxic conditions by flow cytometry. ** P < 0.01, * P < 0.05, *** P < 0.001.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Inhibition, Expressing, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: The knockdown of EZH2 induced the increase of trophoblast autophagy and mTOR pathway inhibition under hypoxic conditions. (A) Significant decreases in EZH2, mTOR and S6K1 mRNA levels were tested in JEG-3 and HTR-8 cells transfected with EZH2 siRNA by qRT-PCR. (B,C) The trophoblast cell lines were transfected with a EZH2 siRNA under hypoxic conditions, and BECN1, P62 and LC3 expression levels were tested by western blotting analysis. Empty vector (scramble) cells served as controls. (D,E) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The two trophoblast cell lines were transfected with EZH2 siRNA with or without AD-MSC exosomes treatment under hypoxic conditions. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Knockdown, Inhibition, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: EZH2 overexpression decreased trophoblast autophagy and proliferation through mTOR signaling pathway. (A) Significant increases in EZH2, mTOR and S6K1 mRNA were found in JEG-3 and HTR-8 cells transfected with EZH2 overexpression plasmid (EZH2) compared to empty vector (Vec)-transfected cells under hypoxic conditions by qRT-PCR. (B,C) The two trophoblast cell lines were transfected with EZH2 overexpression plasmid under hypoxic conditions, and BECN1, P62 and LC3 protein levels were tested by western blotting analysis. (D,E) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The trophoblast cell lines were transfected with a EZH2 overexpression plasmid or empty vector under hypoxic conditions. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: EZH2 regulate trophoblast autophagy and proliferation through mTOR signaling pathway. (A) The levels of EZH2, p-mTOR, p-S6K1, and autophagy associated proteins were examined in JEG-3 and HTR-8 cells transfected with EZH2 overexpression plasmid and treated with m-TOR inhibitor PQR620 under hypoxic conditions by western blot. (B,C) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The trophoblast cell lines were treated with EZH2 overexpression plasmid and m-TOR inhibitor PQR620 under hypoxic conditions. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

doi: 10.3389/fcell.2020.545852

Figure Lengend Snippet: AD-MSC-mediated EZH2 activation increased autophagy in placental explants under hypoxic conditions. (A) Placental explants were treated with AD-MSC exosomes transfected with EZH2 overexpression plasmids or Vec under hypoxic conditions, and EZH2, LC3 expression levels were tested by western blotting analysis. (B) Placental explants were treated with AD-MSC exosomes under hypoxic conditions, and EZH2 were tested by immunohistochemistry. Untreated placental explants served as controls (scale bar, 50 μm). * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human LC3, BECN1, P62, EZH2, mTOR, p-mTOR, S6K1, and p-S6K1 (1:1,000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL, United States) were incubated with the blocked membranes.

Techniques: Activation Assay, Transfection, Over Expression, Expressing, Western Blot, Immunohistochemistry

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: International Journal of Molecular Sciences

Article Title: Role of EZH2 in the Growth of Prostate Cancer Stem Cells Isolated from LNCaP Cells

doi: 10.3390/ijms140611981

Figure Lengend Snippet: LNcap cell line had more CD44 + /CD133 + cells. EZH2 is up-regulated in prostate cancer stem cells (PCSCs) compared with non-PCSCs. ( A ) Identification of the percent of stem cell like cancer cells in two human prostate cancer cell lines by using flow cytometric analysis; ( B ) Histogram of percent of CD44 + /CD133 + cells ( * p < 0.05) in LNcap and PC-3 cell line; ( C ) In all experiments, purity of PCSCs (>90%) was confirmed by flow cytometry. FACS sorted cells were cultured in serum-free medium for 1, 3, 5, 7, 9 days respectively, identification of the percent of CD44 + /CD133 + cells; ( D ) mRNA expression of EZH2 and the putative stem cell markers Oct4, SOX2, Nanog, CXCR4 between PCSCs and non-PCSCs; ( E ) Protein level of of EZH2 and the putative stem cell markers Oct4, SOX2, Nanog, CXCR4 between PCSCs and non-PCSCs.

Article Snippet: Then the membrane was incubated with mouse monoclonal antibody against human EZH2 (Cell Signaling Technology, Danvers, MA, USA) followed by horseradish peroxidase (HRP)-labeled goat-antimouse IgG (Cell Signaling Technology, Danvers, MA, USA) and detected by chemiluminescence.

Techniques: Flow Cytometry, Cell Culture, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role of EZH2 in the Growth of Prostate Cancer Stem Cells Isolated from LNCaP Cells

doi: 10.3390/ijms140611981

Figure Lengend Snippet: ( A ) Protein levels of EZH2 after PCSCs were transfected with siEZH2 and negative control; ( B ) Cell Proliferation ability in PCSCs by MTT assay; ( C – E ) Silence of EZH2 inhibited cell cycle in PCSCs by flow cytometry; ( F ) Histogram of PCSCs cell cycle analysis; ( G – I ) Silence of EZH2 promoted the progression of apoptosis. Apoptosis was analyzed by flow cytometry; ( J ) Histogram of PCSCs apoptosis. All data are from 3 separate experiments ( * p < 0.05).

Article Snippet: Then the membrane was incubated with mouse monoclonal antibody against human EZH2 (Cell Signaling Technology, Danvers, MA, USA) followed by horseradish peroxidase (HRP)-labeled goat-antimouse IgG (Cell Signaling Technology, Danvers, MA, USA) and detected by chemiluminescence.

Techniques: Transfection, Negative Control, MTT Assay, Flow Cytometry, Cell Cycle Assay

Journal: International Journal of Molecular Sciences

Article Title: Role of EZH2 in the Growth of Prostate Cancer Stem Cells Isolated from LNCaP Cells

doi: 10.3390/ijms140611981

Figure Lengend Snippet: ( A ) RNA levels of 8 cell cycle regulators after PCSCs were transfected with siEZH2 and negative control. Cyclin E2 expression decreased significantly after EZH2 knockdown; ( B ) RNA levels of several embryonic development regulators after PCSCs were transfected with siEZH2 and negative control.

Article Snippet: Then the membrane was incubated with mouse monoclonal antibody against human EZH2 (Cell Signaling Technology, Danvers, MA, USA) followed by horseradish peroxidase (HRP)-labeled goat-antimouse IgG (Cell Signaling Technology, Danvers, MA, USA) and detected by chemiluminescence.

Techniques: Transfection, Negative Control, Expressing, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Role of EZH2 in the Growth of Prostate Cancer Stem Cells Isolated from LNCaP Cells

doi: 10.3390/ijms140611981

Figure Lengend Snippet: EZH2 was a direct target of miR-101 in prostate cancer stem cells. ( A ) qPCR analysis of several reported aberrant miRNA which could interact with EZH2, down-regulation of miR-101 was confirmed; ( B ) miR-101 mimic could significantly reduced EZH2 expression compared with controls; ( C ) Luciferase reporter assays in PCSCs: compared with controls, miR-101 mimic group showed a significant difference (* p < 0.05).

Article Snippet: Then the membrane was incubated with mouse monoclonal antibody against human EZH2 (Cell Signaling Technology, Danvers, MA, USA) followed by horseradish peroxidase (HRP)-labeled goat-antimouse IgG (Cell Signaling Technology, Danvers, MA, USA) and detected by chemiluminescence.

Techniques: Expressing, Luciferase