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

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: 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: Early Polycomb-target deregulations in Hutchinson-Gilford Progeria Syndrome revealed by heterochromatin analysis

doi: 10.1101/799668

Figure Lengend Snippet: a , H3K27me3 signal distribution around the TSS region of genes upregulated in HGPS samples compared to controls calculated by deepTools using the genome-wide signal from SPP (see Online methods for details). The x-axis represents relative genomic position around the TSS (+/− 5Kb), and the y-axis represents average signal intensity. b , Overlap of SAMMY-seq domains from each sample to Roadmap Epigenomics chromatin states for normal fibroblasts (E055). The overlaps with each chromatin state are reported as percentage over the total of SAMMY-seq domains (S4 vs S3 or S4 vs S2) for each sample. c , Transcripts differential expression (up-regulation) was assessed by comparing individual HGPS samples against the group of controls, then p-values were aggregated based on their chromatin state, considering only genes in SAMMY-seq domains (S4 vs S2) (see Online methods for details). The plot reports the number of transcripts (x-axis) and significance (Lancaster method aggregated p-values – y-axis) for each chromatin state (color legend) as defined by . d , Representative track of H3K27me3 ChIP-seq signal in 2 control and 3 HGPS samples around the bivalent EN1 gene (chr2: 118810000-118880000), that was upregulated in HGPS samples. e , f , Representative images of PLA experiments in CTRL004 and HGPS167 at late passages (p18). Each fluorescent dot represents the co-localization of Lamin A/C or Progerin and Ezh2 (panel e) or Bmi1 (panel f). Nuclei were stained with dapi. All data were generated from an average of three independent experiments, whiskers represent SEM. g , Western blot on chromatin fractionation experiments of CTRL004 and HGPS167 at early (left) and late (right) passages. Equal amounts of each fraction were hybridized with indicated antibodies. Alpha-tubulin, histone H3 and Lamin A/C were used as loading controls respectively for S1, S2 and S3, S4. h , The graph shows quantifications of Ezh2 and Bmi1 in S4 fraction normalized on S2. Data points were generated from an average of at least two biological replicates, whiskers represent SEM. Comparisons were done using a two-tailed t-test in (e, f, h). Statistically significant differences are marked * p < 0.05; * * p < 0.01.

Article Snippet: The following antibodies were used: Bmi1 (Millipore 05-637, mouse) diluted 1:100; Lamin A/C (Santa Cruz sc-6215, goat) diluted 1:200; Ezh2 (Cell signaling AC22 3147S, mouse) diluted 1:100; H3K9me3 (Abcam ab8898, rabbit) diluted 1:500; H3K27me3 (Millipore 07-449, rabbit) diluted 1:100.

Techniques: Genome Wide, Expressing, ChIP-sequencing, Staining, Generated, Western Blot, Fractionation, Two Tailed Test

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