Journal: Journal of cellular and molecular medicine

Article Title: Increasing RB1 Expression by Targeting EZH2 in Triple-Negative Breast Cancer.

doi: 10.1111/jcmm.70384

Figure Lengend Snippet: FIGURE 2 | The negative correlation between EZH2 and RB1 in TNBC. (A) The Venn diagram illustrates 17 epigenetic regulatory proteins identi- fied from the comparison of 12,301 genes negatively correlated with RB1 versus 167 epigenetic regulatory enzymes (above). The correlation diagram demonstrates that these 17 epigenetic regulatory enzymes exhibit a negative correlation with RB1 (below). (B) Transcriptional analysis from the TCGA database reveals a negative correlation between EZH2 and RB1. Each dot represents a TNBC patient. (C) Western blot analysis of EZH2 and RB1 expression in 15 TNBC cell lines. (D) Quantitative assessment of the results from (C) conducted using ImageJ.

Article Snippet: Primary antibodies used for membrane staining were anti- RB1 (Abcam, ab181616; 1:1000), anti- EZH2 (CST, 5246S; 1:1000), anti- GAPDH (CST, 5174S; 1:1000), anti- H3 (CST, 4499S; 1:1000), anti- H3K27me3 (ABclonal, A19539; 1:1000).

Techniques: Comparison, Western Blot, Expressing

Journal: Journal of cellular and molecular medicine

Article Title: Increasing RB1 Expression by Targeting EZH2 in Triple-Negative Breast Cancer.

doi: 10.1111/jcmm.70384

Figure Lengend Snippet: FIGURE 3 | Inhibiting EZH2 improves RB1 expression. (A, B) Western blot analysis of EZH2, RB1 and H3K27me3 levels in MDA-MB-468 cells following shEZH2 treatment (A) and in HCC1806 cells after CRISPR-KO of EZH2 (B). GAPDH and H3 were utilised as loading controls. (C–F) Western blot analysis of RB1 and H3K27me3 levels in MDA-MB-468 (C), MDA-MB-436 (D), MDA-MB-231 (E) and HCC1806 (F) cells post-treatment with UNC1999 or EPZ6438. GAPDH and H3 served as loading controls. Quantitative analysis of the results was performed using ImageJ. (G–J) The relative mRNA levels of RB1 to GAPDH were determined in (G) MDA-MB-468, (H) MDA-MB-436, (I) MDA-MB-231 and (J) HCC1806 cells post- treatment with UNC1999 or EPZ6438.

Article Snippet: Primary antibodies used for membrane staining were anti- RB1 (Abcam, ab181616; 1:1000), anti- EZH2 (CST, 5246S; 1:1000), anti- GAPDH (CST, 5174S; 1:1000), anti- H3 (CST, 4499S; 1:1000), anti- H3K27me3 (ABclonal, A19539; 1:1000).

Techniques: Expressing, Western Blot, CRISPR

Journal: Journal of cellular and molecular medicine

Article Title: Increasing RB1 Expression by Targeting EZH2 in Triple-Negative Breast Cancer.

doi: 10.1111/jcmm.70384

Figure Lengend Snippet: FIGURE 4 | Inhibiting EZH2 elevates H3K27ac enrichment at the RB1 enhancer region. (A) ATAC-seq tracks the RB1 gene locus in 15 TNBC cell lines, displaying chromatin openness using IgV visualisation software. The cell lines are arranged from top to bottom in descending order of RB1 ex- pression levels, as shown in Figure 2C. (B, C) ChIP-qPCR analysis of H3K27ac at the RB1 enhancer region in MDA-MB-436 (B) or MDA-MB-231 (C) after EPZ6438 treatment. (D) Western blot analysis of RB1 levels in HCC1806 cells with CRISPR knock-out of the RB1 enhancer region. (E) Relative mRNA levels of RB1 to GAPDH in HCC1806 cells with CRISPR knock-out of the RB1 enhancer region.

Article Snippet: Primary antibodies used for membrane staining were anti- RB1 (Abcam, ab181616; 1:1000), anti- EZH2 (CST, 5246S; 1:1000), anti- GAPDH (CST, 5174S; 1:1000), anti- H3 (CST, 4499S; 1:1000), anti- H3K27me3 (ABclonal, A19539; 1:1000).

Techniques: Software, ChIP-qPCR, Western Blot, CRISPR, Knock-Out

Journal: PLoS ONE

Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

doi: 10.1371/journal.pone.0027596

Figure Lengend Snippet: (A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: ChIP assays were performed as previously described (Tong Ihn Lee, 2006) using 5 μg of antibodies against YY1 (rabbit polyclonal from Santa Cruz Biotechnology, Cat# SC-1703), Ezh2 (mouse monoclonal from Cell Signaling, Cat# AC22), trimethyl-histone H3-K27 (rabbit polyclonal from Milipore, Cat# 07-449), or without any antibody as a negative control.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Control, Expressing, Cell Culture, Luciferase, Activity Assay, Negative Control, Reporter Assay, Over Expression

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: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a EZH2 ChIP peak profiles of gene Otx2 for WT, Mtf2 null, and Jarid2 null cells. Peaks were RPKM normalized and scaled across backgrounds. b Phase-contrast of Embryoid Bodies from different background and time point of differentiation. Each differentiation time point and background were performed in replicates. c , d Single-cell UMAPs of 4949 cells over all backgrounds and time points. e Single-cell UMAP of cells colored by predicted clusters. f – i Feature maps of selected lineage genes, color intensity based on normalized expression of individual gene.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Heatmap of differentially expressed genes comparing different mutants against WT in undifferentiated mESCs. Z-score normalized counts (row scaling) are shown in heatmap. b Venn diagram depicting overlap of a number of genes which were up/downregulated in Mtf2 null and Jarid2 null cells, with PRC2 targets (as determined from EZH2 ChIP targets; next panel). Significance of overlap calculated using hyper-geometrical test. c Heatmap of EZH2 binding peaks (RPKM normalized) for upregulated genes (from Mtf2 and Jarid2 null ESCs) for different genetic backgrounds. Heatmap depicts a window of +/−400 bp from the transcription start site (TSS) of the genes. d Dot plot showing the enriched Gene Ontology terms for the differentially expressed genes in different genetic backgrounds.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a ChIP peak profiles of histone mark H3K27me3 and H3K4me3 for selected upregulated ( Mtf2 null) lineage transcription factors for WT and Mtf2 null cells at pluripotent stage. ChIP profiles were RPKM normalized and scaled between two merged profiles per histone ChIP. b Boxplots depicting the RPKM values of promoter (as defined by +/−500 bp from TSS) H3K27me3, H3K4me3, and EZH2 for all PRC2 targets and the upregulated genes in Mtf2 null and Jarid2 null cells. Asterisk(*) represents a Two-sample Kolmogorov–Smirnov test p -value <0.05. n = 2878 for all PRC2 targets, n = 242 for upregulated Mtf2 null genes and n = 58 for upregulated Jarid2 null genes. Whisker ends of boxplot represent the maximum (top) and minimum values, respectively. Top and bottom of boxplots represent 75th and 25th percentile values, respectively, and finally, median values are shown as colored lines within the boxplots. c Transcription factor motif activity that explains part of the variance in transcript levels, based on motifs in the promoters (as defined by +/−500 bp from TSS) of all upregulated PRC2-bound genes (“Methods”; upregulated genes in all four cell lines, cf. Fig. ). Motifs are shown in aggregated z-scores. d – f Heatmaps showing the mRNA fold change, H3K27me3 and H3K4me3 levels for a set of transcription factors (left, identified in panel c ) and signaling factors (right, identified from Mtf2 and Jarid2 null DEGs list). Genes shown are all PRC2 targets. g Barplot depicting the GATA2 ChIP recovery relative to input. Control is a gene desert region (“Methods”). Dots in bars represent 4 replicates per sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Whisker Assay, Activity Assay, Control