Journal: Journal of Veterinary Research

Article Title: Clinicopathological Analysis of Expression of Enhancer of Zeste Homologue 2 in Canine Mammary Carcinoma

doi: 10.2478/jvetres-2022-0033

Figure Lengend Snippet: Transcription level of the EZH2 gene in canine normal mammary gland and CMC tissues A – normal canine mammary tissues; B – infiltrating ductal carcinoma; C – intraductal papillary carcinomas; D – infiltrating micropapillary carcinoma; E – ductal carcinoma in situ Results are presented as mean ± SD of tissues in the group. * P < 0.05 and ** P < 0.01 show significantly different by one ANOVA test

Article Snippet: Rabbit monoclonal anti-EZH2 antibody (bsm-60001R) was obtained from Bioss (Beijing, China) and used at a dilution of 1:100.

Techniques: In Situ

Journal: Journal of Veterinary Research

Article Title: Clinicopathological Analysis of Expression of Enhancer of Zeste Homologue 2 in Canine Mammary Carcinoma

doi: 10.2478/jvetres-2022-0033

Figure Lengend Snippet: Expression of the EZH2 protein in normal canine mammary gland and CMC tissue as revealed by immunohistochemical staining (200 ×) A – normal canine mammary tissues; B – infiltrating ductal carcinoma; C – intraductal papillary carcinomas; D – infiltrating micropapillary carcinoma; E – ductal carcinoma in situ . Scale bars: 50 μm

Article Snippet: Rabbit monoclonal anti-EZH2 antibody (bsm-60001R) was obtained from Bioss (Beijing, China) and used at a dilution of 1:100.

Techniques: Expressing, Immunohistochemical staining, Staining, In Situ

Journal: Journal of Veterinary Research

Article Title: Clinicopathological Analysis of Expression of Enhancer of Zeste Homologue 2 in Canine Mammary Carcinoma

doi: 10.2478/jvetres-2022-0033

Figure Lengend Snippet: Relationship between expression of EZH2 and clinicopathological factors in CMCs

Article Snippet: Rabbit monoclonal anti-EZH2 antibody (bsm-60001R) was obtained from Bioss (Beijing, China) and used at a dilution of 1:100.

Techniques: Expressing, In Situ

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 1 | M2 macrophage polarization in patients with glioma is associated with EZH2 overexpression. (A) Immunohistochemistry analysis of EZH2 in clinical samples of different grades of gliomas (×200). (B) Immunohistochemistry analysis of CD206 in clinical samples of different grades of gliomas (×200). (C) EZH2 and CD206 immunohistochemical scores of clinical specimens of different grades of gliomas. (D) Pearson correlation analysis of EZH2 and CD206 immunohistochemical scores in glioma clinical samples. (E) EZH2 expression in glioma clinical specimens determined by RT-qPCR. (F) The expression of IL-6 in glioma clinical specimens determined by RT-qPCR. (G) The expression of IL-8 in glioma clinical specimens determined by RT-qPCR. (H) The expression of MIP-3α in glioma clinical specimens determined by RT-qPCR. n = 30 in WHO II group; n = 30 in WHO III group; n = 30 in WHO IV group; n = 30 in normal group. *p < 0.05 vs. normal brain tissues. #p < 0.05 vs. WHO II glioma tissues. &p < 0.05 vs. WHO III glioma tissues. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Pearson correlation analysis was performed to observe the correlation of indicators.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Over Expression, Immunohistochemistry, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 2 | The growth of glioma and M2 macrophage polarization is repressed by silencing EZH2 in nude mice. (A) The successful establishment of glioma xenograft model in nude mice determined using HE staining (×200). (B) Western blots of EZH2 protein. (C) Western blot analysis to verify the silencing efficiency of Lenti-EZH2. (D) The expression of N-cadherin and Vimentin proteins measured by Western blots. (E) A172 cells treated with Lenti-EZH2 or Lenti-HK were co-injected with polarized macrophages into the axilla of nude mice, and the size of glioma was measured and analyzed 2 months later. (F) Tumor size of mice. (G) The GFP fluorescence intensity of glioma formation in nude mice measured by an in vivo imaging system (IVIS spectrum), and the signal intensity of ROI reflected the growth of the tumor. (H) Statistical analysis of ROI signal intensity. (I) Immunofluorescence detection of the expression of CD206, MIP-3α, IL-6, and IL-8 in tumor tissues of mice (×400). (J) Statistical analysis of immunofluorescence intensity of CD206, MIP-3α, IL-6, and IL-8 in mice. (K) The expression of MIP-3α, IL-6, and IL-8 in plasma of nude mice evaluated by ELISA. *p < 0.05 vs. nude mice injected with Lenti-HK-treated glioma cells and polarized macrophages. n = 8. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Staining, Western Blot, Expressing, Injection, In Vivo Imaging, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 3 | Downregulated EZH2 alters the microenvironment of glioma and suppresses the polarization of M2 macrophages co-cultured in vitro. (A) The proportion of CD11b+ CD206+ cells in the co-cultured cells determined by flow cytometry. (B) The expression of IL-8, MIP-3α, and IL-6 in macrophages measured by RT-qPCR. (C) The expression of IL-8, IL-6, and MIP-3α in macrophages detected by immunofluorescence (×400). (D) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3α in macrophages. (E) The content of IL-8, MIP-3α, and IL-6 in cell culture supernatant detected by ELISA. (F) The expression of M1 polarization markers II1b, Cd86, and Nos2 as well as M2 markers Cd163, Ym1, and Mrc1 in macrophages determined by RT-qPCR. *p < 0.05 vs. treatment with Lenti-HK. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test. Experiments were repeated three times independently.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Cell Culture, In Vitro, Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 4 | EZH2 contributes to miR-454-3p downregulation via DNA methylation to promote polarization of M2 macrophages. (A) The expression of miR-454-3p in clinical samples of different grades of glioma detected by RT-PCR. *p < 0.05 vs. normal brain tissues, #p < 0.05 vs. WHO II glioma tissues, &p < 0.05 vs. WHO III glioma tissues. (B) The efficiency of miR-454-3p and miRZIP-454 verified by RT-qPCR. *p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (C) Pearson correlation analysis of EZH2 and miR-454-3p expression in glioma clinical samples. (D) RT-qPCR to detect the expression of miR-454-3p and PTEN in glioma cells after EZH2 inhibition. *p < 0.05 vs. the treatment with Lenti-HK. (E) The proportion of CD11b+ CD206+ cells in cells after co-culture of miR-454-3p-overexpressed/knockout A172 cells with THP-1 cells detected by flow cytometry. (F) The expression of IL-8, IL-6, and MIP-3α in the macrophages after co-culture with A172 cells treated with Lenti-PTEN determined by RT-qPCR. (G) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3α in the tumor microenvironment. (H) The levels of IL-8, IL-6, and MIP-3α in the supernatant detected by ELISA. p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (I) CpG island and primer prediction. (J) MethPrime analysis of promoter CpG islands of miR-454-3p. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Pearson correlation analysis was performed to observe the correlation of indicators. Experiments were repeated three times independently.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: DNA Methylation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Control, Inhibition, Co-Culture Assay, Knock-Out, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 7 | The scheme of the mechanism by which EZH2 affects glioma tumorigensis. EZH2 inhibits miR-454-3p to enhance the binding to m6A reading protein YTHDF2, whereby promoting m6A modification of PTEN and inducing M2 macrophage polarization in glioma and tumorigensis.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Maternal obesity alters histone modifications mediated by the interaction between EZH2 and AMPK, impairing neural differentiation in the developing embryonic brain cortex

doi: 10.1016/j.jbc.2025.108173

Figure Lengend Snippet: Maternal obesity affects Ezh2 and alters global histone modifications in the embryo brain cortex. A , Western blot analysis for expressing Ezh2-Thr311p, Ezh2-Thr487p, and total Ezh2 on control and HFD embryo brain cortical tissue lysates at E14.5, E16.5 and E18.5 stages. The expression of β-Actin was used as the loading control. B , densitometric quantitation of Western blots from panel A . C , Western blot analysis for the expression of histone modifications, H3K27me3, H3K4me4, H3K4me2, pan H3, H4K16Ac, and pan H4 on control and HFD embryo brain cortical tissue lysates at E14.5, E16.5, and E18.5 stages. The expression of β-Actin was used as the loading control. D , densitometric quantitation of Western blots from panel C . n = 3. The data ( bars ) are represented as mean ± SD. ∗∗ p < 0.01 and ∗ p < 0.05. n/s, not significant; HFD, high-fat diet.

Article Snippet: Phospho EZH2 (Thr 311) , Cell Signaling Technology , 27888 , Western blotting, ChIP.

Techniques: Western Blot, Expressing, Control, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Maternal obesity alters histone modifications mediated by the interaction between EZH2 and AMPK, impairing neural differentiation in the developing embryonic brain cortex

doi: 10.1016/j.jbc.2025.108173

Figure Lengend Snippet: Maternal obesity is associated with reduced H3K27me3 levels on gene promoters, contributing to transcriptional derepression. A , heat map of ChIp-seq data showing enrichments at −2500 bp to +2500 bp around the transcription start site (TSS) for histone H3 and H4 and modifications, H3K27me3, H3K4me3, and H4K16Ac in control and HFD embryo brain cortices from E14.5. B , line plots for ChIP-seq enriched peaks at −2500 bp to +2500 bp around the TSS (H3K27me3, H3K4me3, H3, and H4K16Ac, H4) on the list of genes upregulated in RNA-seq in HFD embryo brain cortices compared to control at E14.5. C , ChIP-seq peaks for H3K27me3, H3K4me3, pan H3 correlation with gene expression changes ( red arrow = upregulation; green arrow = downregulation) observed in RNA-seq for four genes, Bmp4 , Wnt1 , Pdgfra , and Ccn3 in control and HFD embryo brain cortices from E14.5. D , ChIP-seq peaks for H3K27me3, H3K4me3, pan H3 correlation with gene expression changes (upregulation) observed in RNA-seq for four noncortical TFs genes, Pax9 , Six1 , Twist1 , and Dlx5 in control and HFD embryo brain cortices from E14.5. E , ChIP-qPCR was done to analyze the enrichment of pan H4, H4K16Ac, pan H3, H3K27me3, and H3K4me3 at the promoter (−500 to +500 bp of TSS) of six upregulated genes, Ccn3 , Pgdfra , Bmp4 , Twist1 , Pax9 , and Runx2 in control and HFD embryo brain cortices at E14.5. ChIP with IgG was used as control. Input is the total DNA. F , ChIP-qPCR to analyze the enrichment of the Ezh2, Ezh2-Thr311p, and Ezh2-Thr487p at the promoter (−500 to +500 bp of TSS) of six upregulated genes, Ccn3 , Pgdfra , Bmp4 , Twist1 , Pax9 , and Runx2 in control and HFD embryo brain cortices from E14.5. ChIP with IgG was used as a control. Input is the total DNA. n = 3. The data ( bars ) are represented as mean ± SD. ∗∗ p < 0.01 and ∗ p < 0.05. n/s, not significant; HFD, high-fat diet; qPCR, quantitative polymerase chain reaction; TF, transcription factor; ChIP-seq, chromatin immunoprecipitation sequencing.

Article Snippet: Phospho EZH2 (Thr 311) , Cell Signaling Technology , 27888 , Western blotting, ChIP.

Techniques: ChIP-sequencing, Control, RNA Sequencing, Gene Expression, ChIP-qPCR, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Maternal obesity alters histone modifications mediated by the interaction between EZH2 and AMPK, impairing neural differentiation in the developing embryonic brain cortex

doi: 10.1016/j.jbc.2025.108173

Figure Lengend Snippet: Ampk-Thr172p and Ezh2 interaction is increased in HFD. A , Western blot analysis for the expression of Ampk and Ampk-Thr172p on control and HFD embryo brain cortical tissue lysates at E14.5, E16.5, and E18.5 stages. The expression of β-Actin (same as in Fig. 5 A as the same samples were analyzed in both figures) was used as the loading control. B , densitometric quantitation of Western blots from panel A . C , coimmunoprecipitation was done on cell lysates from Ctrl and HFD embryo brain cortical tissue lysates from E14.5 using anti-Ezh2 antibody, followed by Western blotting of IP’d samples using anti-Ampk and Ampk-Thr172p antibodies. IP with IgG was used as a control. Input is the total cell lysate. D , densitometric quantitation of Western blots from panel C . E , Western blot analysis for the expression of global O-GlcNAc levels on control and HFD embryo brain cortical tissue lysates at E14.5, E16.5, and E18.5 stages. The expression of β-Actin was used as the loading control. F , densitometric quantitation of Western blots from panel E . G , coimmunoprecipitation was done on cell lysates from Ctrl and HFD embryo brain cortical tissue lysates from E14.5 anti-O-GlcNAc antibody, followed by Western blotting for Ezh2, Ezh2-Thr311p, Ampk, and AMPK-Thr172P. IP with IgG was used as a control. Input is the total cell lysate. H , densitometric quantitation of Western blots from panels G . Data represents the mean of three biological replicates ± SD. HFD, high-fat diet; IP, immunoprecipitation.

Article Snippet: Phospho EZH2 (Thr 311) , Cell Signaling Technology , 27888 , Western blotting, ChIP.

Techniques: Western Blot, Expressing, Control, Quantitation Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Maternal obesity alters histone modifications mediated by the interaction between EZH2 and AMPK, impairing neural differentiation in the developing embryonic brain cortex

doi: 10.1016/j.jbc.2025.108173

Figure Lengend Snippet: List of Antibodies used for Western blotting. immunohistochemistry, ChIP, and ChIP-seq

Article Snippet: Phospho EZH2 (Thr 311) , Cell Signaling Technology , 27888 , Western blotting, ChIP.

Techniques: Western Blot, Immunohistochemistry, Purification

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The primers used for RT-qPCR.

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Sequencing

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The sphere formation ability of the RCC cell lines and the expression of potential CSC markers on the mRNA and protein levels. ( A ) Sphere formation: SKRC-17 and RCC-53 formed spheres around day 7, RCC-26 did not. Photos were taken by a microscope digital camera at the magnification of 100× (Bresser GmbH DE-46414 Rhede Germany). ( B ) mRNA expression of 19 potential CSC markers in SKRC-17, RCC-53, and their corresponding CSCs analyzed by RT-qPCR analysis ( p < 0.05 indicates statistical significance), the expression level observed in spheres was normalized to the corresponding adherent line. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( C ) Representative images of IHC staining for the EZH2, ABCG2, ALDH1A3, and SALL4 expression in the KIRC and normal tissue, photos derived from the Human Protein Atlas database. ( D ) Measurement of the protein expression in the adherent and sphere cell lines by flow cytometry ( n = 3, adherent cell value was set as 1).

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Immunohistochemistry, Derivative Assay, Flow Cytometry

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The correlation of EZH2 expression with clinicopathological characteristics: ( A ) Kaplan–Meier survival analysis for the KIRC patients grouped into high or low score in EZH2 expression determined by the comparison with the median, p < 0.001 by log-rank test. ( B ) Distribution of EZH2 expression concerning the stage, grade, and T classification, the p -values p = 0.003, 0.022, and 0.002, respectively, were calculated by the Kruskal–Wallis rank sum test, distribution of scores in M and N classification, the p -values p = 0.001 and 0.0015, respectively, were calculated by the Wilcoxon rank sum test. ( C ) The multivariate Cox regression analysis of the risk score, age, gender, grade, and TNM stage was used to evaluate the independent prognostic value of EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Expressing

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The correlation of the TIC composition and signaling pathway with EZH2 expression. ( A ) Correlation of the TIC population with EZH2 expression, the Violin diagram shows the relationship between the different 22 TIC subpopulations and the KIRC tumor samples with low (green) or high (red) EZH2 expression, relative to the median of the EZH2 expression level, the Wilcoxon rank sum was used for the significance test. ( B ) The scatter plot shows the correlation of 12 TIC subpopulations to the EZH2 expression ( p < 0.05), the blue line in each plot shows the fitted linear model indicating the proportion tropism of the immune cell along with the EZH2 expression, and the Pearson coefficient was used for the correlation test. ( C ) The Venn plot displays nine TIC subpopulations correlated with the EZH2 expression codetermined by difference and correlation tests displayed in violin and scatter plots, respectively. ( D ) The gene set enrichment analysis for the enriched gene sets with high and low EZH2 expression in the KEGG collection, each line represents one particular gene set with a unique color, upregulated gene sets are located on the left near at the origin of the coordinates, in contrast, the downregulated gene sets are on the right of the x -axis. Only gene sets with NOM p < 0.05 and FDR q < 0.06 were considered as significant and only some of the leading pathways are displayed in the plot.

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Expressing

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: EGCG inhibited the expression of EZH2 in ccRCC CSCs. ( A ) CellTiter-Blue Cell Viability Assay: EGCG, wogonin, apigenin, and shikonin inhibited the viability of SKRC-17, RCC-53 in a dose-dependent manner, shown after 24 and 48 h. The half-maximal inhibitory concentration (IC50) was calculated by the logit regression model. ( B ) RT-qPCR analysis: EGCG, wogonin, apigenin, and shikonin reduced the expression of EZH2 in the sphere cells, normalized expression levels are displayed ( n = 3, * p < 0.05, **** p < 0.0001).

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Expressing, Viability Assay, Concentration Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The interaction network based on the potential targets of EGCG. ( A ) The PPI network of potential target genes of EGCG and EZH2; the edges represent the predicted functional associations, green line: the neighborhood evidence, blue line: co-occurrence evidence, purple line: experimental evidence, yellow line: text mining evidence, black line: co-expression evidence, dotted lines: the potential target genes of EGCG and EZH2. ( B ) The network of EGCG, EZH2 target genes, and EZH2, green lines represent the interaction between EGCG and its target genes, red lines represent interaction between EZH2 and target genes, black lines represent interaction among target genes.

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques: Functional Assay, Expressing

Journal: Cancers

Article Title: Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG)

doi: 10.3390/cancers14174200

Figure Lengend Snippet: The proposed model for the correlation among EZH2, cancer stem cells, and EGCG for the KIRC patients.

Article Snippet: Spheres and adherent tumor cell lines were harvested and stained with the following mouse monoclonal antibodies: EZH2 (clone 11/EZH2 Alexa Fluor ® 647, BD Biosciences, Heidelberg, Germany), ALDH1A3 (clone OTI4E8, ORIGENE, Rockville, MD, USA), SALL4 (clone 6E3, Abcam, Cambridge, UK), and ABCG2 (clone 5D3/CD338, APC, BD Biosciences), respectively.

Techniques:

Journal: Cell Death & Disease

Article Title: Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2

doi: 10.1038/s41419-019-1742-7

Figure Lengend Snippet: a Western blotting analysis showing that Neat1 knockdown enhanced P21 protein expression but decreased Cdk4 protein expression in C2C12 cells. The relative protein levels of P21 and Cdk4 were quantified using ImageJ software. b , c ChIP-qPCR results revealed that the enrichments of Ezh2 ( b ) and H3k27me3 ( c ) at the P21 promoter were significantly decreased after Neat1 knockdown. d , e ChIP-qPCR results revealed that the enrichments of Ezh2 ( d ) and H3k27me3 ( e ) at the P21 promoter were significantly increased after Neat1 overexpression. f Co-transfection of Ezh2 siRNA fragment and Neat1 expression vector in C2C12 cells for 2 days. Immunofluorescence staining of P21 was performed, and P21 expression was quantified by ImageJ. The quantification of P21 immunofluorescence staining results showed that the overexpression of Neat1 inhibited P21 protein expression, but had no significant effect on P21 expression after co-transfection with Ezh2 siRNA fragment. g Neat1 siRNA fragment and Ezh2 expression vector were co-transfected into C2C12 cells and the cells were performed with EdU staining at 2 days after transfection. The percentage of EdU + cells was quantified. The quantification of EdU staining results showed that Neat1 knockdown inhibited myoblast proliferation. After co-transfected with Ezh2 expression vector, Neat1 knockdown can not inhibit myoblast proliferation. h Neat1 and P21 siRNA fragments were co-transfected into C2C12 cells and the cells were performed with EdU staining at 2 days after transfection. The quantification of EdU staining results showed that Neat1 knockdown significantly reduced the percentage of EdU + cells, but did not reduce the number of EdU + cells after co-transfection with P21 siRNA fragment. Protein levels were normalized to those of β-actin. All values represent the mean ± s.d. of three independent experiments. * p < 0.05, ** p < 0.01, N.S. indicates not significant

Article Snippet: The antibodies and their dilutions were shown as following: Myod (Santa Cruz Biotechnology, USA; sc-760; 1:1000), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:200), Myhc (Santa Cruz Biotechnology, USA; sc-376157; 1:3000), α-actin (Proteintech, China; 23660-1-AP; 1:1000), Tnni2 (Abcam, UK; ab184554; 1:1000), P21 (BOSTER, China; BM4382; 1:200), Ezh2 (Cell Signaling Technology, USA; 5246; 1:1000), Pcna (Servicebio, China; GB11010; 1:500), Ki67 (Abcam, UK; ab16667; 1:1000), β-actin (Santa Cruz Biotechnology, USA; sc-4777; 1:1000), Gapdh (BOSTER, China; BM3876; 1:200), Pax7 (Developmental Studies Hybridoma Bank; USA; 1:1000), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:1000).

Techniques: Western Blot, Knockdown, Expressing, Software, ChIP-qPCR, Over Expression, Cotransfection, Plasmid Preparation, Immunofluorescence, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2

doi: 10.1038/s41419-019-1742-7

Figure Lengend Snippet: In proliferating myoblasts, Neat1 guides Ezh2 to the P21 promoter and inhibits P21 expression, leading to the promotion of myoblast proliferation. Upon differentiation, Neat1 recruits Ezh2 to inhibit the expression of muscle-specific genes, such as Myog , Myh4 , and Tnni2 , and suppresses myogenic differentiation

Article Snippet: The antibodies and their dilutions were shown as following: Myod (Santa Cruz Biotechnology, USA; sc-760; 1:1000), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:200), Myhc (Santa Cruz Biotechnology, USA; sc-376157; 1:3000), α-actin (Proteintech, China; 23660-1-AP; 1:1000), Tnni2 (Abcam, UK; ab184554; 1:1000), P21 (BOSTER, China; BM4382; 1:200), Ezh2 (Cell Signaling Technology, USA; 5246; 1:1000), Pcna (Servicebio, China; GB11010; 1:500), Ki67 (Abcam, UK; ab16667; 1:1000), β-actin (Santa Cruz Biotechnology, USA; sc-4777; 1:1000), Gapdh (BOSTER, China; BM3876; 1:200), Pax7 (Developmental Studies Hybridoma Bank; USA; 1:1000), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:1000).

Techniques: Expressing