Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images showing the expression of EZH2, SUZ12 and H3K27me3 in cultured primary cortical neurons. Yellow arrowheads indicate Tuj1 positive neurons. Scale bar, 50µm. (b) Top: representative images of P0 mouse coronal brain section stained with anti-Cre antibody showing the expression of Cre at cortical and hippocampal region. Bottom: the enlarged images of two white dashed boxes shown in the top panel. Scale bar, 500µm in the bottom panel and 2 mm in the top panel. (c) NeuroD6-Cre mediated recombination in the adult mouse brain was confirmed by RT-PCR. Representative PCR band image showing a 254bp fragment generated by NeuroD6-Cre mediated Ezh2 deletion.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Expressing, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Generated

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images showing the deletion of EZH2 in Cre-positive hippocampal neurons of Ezh2 Δ/Δ mice in vitro . detected by immunostaining of EZH2. The white arrows indicate the Tuj1 positive neurons. Scale bar, 20µm. (b) Quantification of the fluorescence intensity of EZH2 staining shown in (a). n=74 and 91 for the control and Ezh2 Δ/Δ neurons, respectively, from three independent experiments. **** P <0.0001. (c) Representative images showing reduced level of H3K27me3 expression in Cre-positive hippocampal neurons in vitro . The white arrows in the upper panel indicate Cre negative neurons from the control mice. The white arrows in the bottom panel indicate the Cre positive neurons from the Ezh2 Δ/Δ mice, whereas the red arrow indicates a Cre negative neuron. Scale bar, 20µm. (d) Quantification of the fluorescence intensity of H3K27me3 staining shown in (c). n=45 and 37 for the control and Ezh2 Δ/Δ neurons, respectively, from three independent experiments. **** P <0.0001. (e) Representative images showing reduced level of H3K27me3 in cortical neurons from cortical slices of Ezh2 Δ/Δ mice in vivo by immunostaining of H3K27me3. The white arrows in the bottom panel indicate the Cre positive neurons in the Ezh2 Δ/Δ cortical slices, and the red arrow indicates a Cre negative neuron. Scale bar, 20µm. (f) Quantification of the fluorescence intensity of H3K27me3 staining shown in (e). n=103 and 111 neurons for the control and Ezh2 Δ/Δ neurons, respectively, from three independent experiments. **** P <0.0001. (g) Representative western blot images showing significant reduction of EZH2 protein levels in cortical lysate from Ezh2 Δ/Δ mice compared to control at P0. Histone 3 (H3) was used as the loading control. (h) Quantification of western blot results shown in (g) by measuring the ratio of EZH2 and H3, and the data were normalized to the control ( P = 0.0117, n=5 mice for each condition). Data are represented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: In Vitro, Immunostaining, Fluorescence, Staining, Expressing, In Vivo, Western Blot

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative nissl staining images of coronal sections from P0 control and Ezh2 Δ/Δ mice showing no obvious difference between the control and the Ezh2 Δ/Δ mice. (b) Representative images of coronal brain sections stained with anti-Tbr1 antibody and DAPI showing that Ezh2 Δ/Δ mice have normal early neurogenesis at E14.5. Scale bar, 100µm

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Staining

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images of mouse cortex immunostained for Foxp1 and Ctip2 in control and EZH2 Δ/Δ mice at P0. Foxp1 and Ctip2 labeling help to define the layer II-IV and IV-V of the cortical plate, respectively. The distributions of labeled cells were assigned into 6 bins across the apicobasal axis. Scale bar, 100 µm. (b) Quantification of Foxp1 positive cells in 6 equal bins showing significantly reduced Foxp1 positive neurons in Bin 5 (Bin1, P =0.2495; Bin2, P =0.1263; Bin3, P =0.9192; Bin4, P =0.5790; Bin5, P =0.0213, n=3 mice for each condition). (c) Quantification of Ctip2 positive cells in 6 equal bins showing significantly increased Ctip2 positive neurons in Bin 3 (Bin1, P =0.1885; Bin2, P =0.0822; Bin3, P =0.016; Bin4, P =0.8954, n=3 mice for each condition). (d) Representative confocal images of EZH2 f/f mice mouse cortices in utero electroporated with dsRed or dsRed/ Neurod1-Cre . The electroporation was done at E15 and pups were sacrificed at P0 for analysis. The two white dashed line boxes in the upper panels were enlarged and presented in the bottom panels. Scale bar, 200 µm in the upper panels and 50 µm in the bottom panels. (e) Quantification of dsRed positive cells in mouse cortices of (d) showing significantly increased number of cells at Bin1 but reduced number of cells at Bin6 in mice electroporated with Neurod1-Cre (Bin1, P =0.0325; Bin2, P =0.0019; Bin3, P =0.1375; Bin4, P =0.2140; Bin5, P =0.2502; Bin6, P =0.0016; n=3 mice for each condition). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Labeling, In Utero, Electroporation

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images of cultured hippocampal neurons from control and Ezh2 Δ/Δ mice stained with Cre and MAP2 antibodies. Scale bar, 20 µm. (b) Representative images of several MAP2 stained hippocampal neurons from control and Ezh2 Δ/Δ mice. Note the increased dendritic branching of neurons from the Ezh2 Δ/Δ mice. Scale bar, 20 µm. (c) Sholl analysis of hippocampal neurons from control and EZH2 Δ/Δ mice showing that, compared with that of the control mice, the neurons of the EZH2 Δ/Δ mice had increased dendritic branching. ( P =0.2759 at 10µm; P =0. 0.0006 at 20 µm; P =0.0006 at 30 µm; P =0.0004 at 40 µm; P = 0. 0.0023 at 50 µm; P = 0.0006 at 60 µm; P = 0.0174 at 70 µm; P = 0.0757 at 80 µm; P = 0.0078 at 90 µm; P = 0.0007 at 100 µm; P = 0.0012 at 110 µm; n=30 and 32 neurons of control and Ezh2 Δ/Δ mice, respectively, each from 3 to 4 mice). (d) Quantification of normalized average dendritic branch length (ADBL) of control and EZH2 Δ/Δ neurons ( P = 0.0441, n = 35 and 32 neurons from the control and the Ezh2 Δ/Δ mice, respectively, from 3 independent experiments). (e) Quantification of normalized total dendritic branch tip number (TDBTN) of control and EZH2 Δ/Δ neurons ( P < 0.0001, n = 35 and 32 neurons from the control and the Ezh2 Δ/Δ mice, respectively, from 3 independent experiments). (f) Quantification of normalized total dendritic branch length (TDBL) of control and EZH2 Δ/Δ neurons (P <0.0001, n = 35 and 32 neurons from the control and the Ezh2 Δ/Δ mice, respectively, from 3 independent experiments). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Cell Culture, Staining

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images of Ezh2 Δ/Δ ; Thy1-GFP mice. GFP expression was detected at cortical and hippocampal region. SS indicates the somatosensory cortex and CA1 indicates the CA1 region of the hippocampus. Scale bar, 1 mm. (b) Representative images of cortical neurons in Ezh2 f/f ; Thy1-GFP and Ezh2 Δ/Δ ; Thy1-GFP mice. Scale bar, 20µm (c) Representative drawing of neurons in Ezh2 f/f ; Thy1-GFP (left panel) and Ezh2 Δ/Δ ; Thy1-GFP (middle panel) mice. The orange colored are basal dendrites and the blue colored are apical dendrites. The right panel shows a representative image of a GFP labeled cortical neuron. Scale bar, 20µm (d) Sholl analysis for apical dendrites of cortical neurons showing no significant difference in dendritic trees between control and Ezh2 Δ/Δ cortical neurons ( n = 14 neurons from 4 mice for each condition). (e) Sholl analysis for basal dendrites of cortical neurons showing significantly increased complexity of Ezh2 Δ/Δ cortical neurons, compared with that of control mice ( P = 0.03 at 25µm, n=14 neurons from 4 mice for each condition). (f) Quantification of normalized average dendritic branch length (ADBL) of control and Ezh2 Δ/Δ neurons in vivo ( P =0.0014, n=18 and 17 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 mice from each condition). (g) Quantification of normalized total dendritic branch tip number (TDBTN) of control and Ezh2 Δ/Δ neurons in vivo ( P =0.0211, n=18 and 17 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 mice from each condition). (h) Quantification of normalized total dendritic branch length (TDBL) of control and Ezh2 Δ/Δ neurons in vivo ( P =0.0879, n=18 and 17 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 mice from each condition). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Expressing, Labeling, In Vivo

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images of increased spine density in basal region of hippocampal neurons from control and Ezh2 Δ/Δ mice. The yellow images are white images processed with the software iMaris. (b) Quantification of spine density in the basal region of hippocampal neurons from control and Ezh2 Δ/Δ mice ( P =0.0260, n=28 and 24 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 mice for each condition). (c) Left: quantification analysis of different spine types in basal region of hippocampal neurons from control and Ezh2 Δ/Δ mice ( P =0.0003 for mushroom; P =6.86624E-05 for filophodia; n=15 and 11 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 mice for each condition. Right: diagram showing 4 different spine types. (d) Representative traces of mEPSCs of hippocampal neurons from control and Ezh2 Δ/Δ mice (e) Quantification of average frequency and amplitude of mEPSCs of hippocampal neurons from control and Ezh2 Δ/Δ mice (Frequency: P =0.0348; Amplitude: P =0.8299, n=11 and 15 neurons for the control and the Ezh2 Δ/Δ mice, respectively, from 4 independent experiments for each condition). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Software

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative images of increased spine density in basal region of layer IV-V neurons from Ezh2 f/f ; Thy1-GFP and Ezh2 Δ/Δ ; Thy1-GFP mice. (b) Quantification analysis of spine density in deep region of cortical neurons from control and Ezh2 Δ/Δ mice. n=25 for control mice and n=26 for Ezh2 Δ/Δ mice, P =0.0002. (c) Representative images of increased spine density in basal region of layer II-III neurons from Ezh2 f/f ; and Ezh2 Δ/Δ mice labeled with Golgi staining. (d) Quantification analysis of spine density in layer II-III region of cortical neurons from control and Ezh2 Δ/Δ mice. n=25 for control mice and n=26 for Ezh2 Δ/Δ mice, P =0.0095. Data are presented as mean ± SEM. n.s., no significant difference, * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Workflow of collecting excitatory neurons from control, Nestin-Cre; Ezh2 f/f , and EZH2 Δ/Δ mice for RNA-seq analysis. (b) Gene ontology analysis of upregulated and downregulated differential expressed genes in Nestin-Cre; Ezh2 f/f and EZH2 Δ/ Δ mice, compared to that of the control mice. (c) Venn diagram representing the number of DEGs between SFARI autism genes and Nestin-Cre; Ezh2 f/f or EZH2 Δ/Δ mice. (d) Quantitative Reverse Transcription PCR analysis of selected DEGs in excitatory neurons in wildtype and EZH2 Δ/Δ mice ( P = 0.02 for PAK3, P = 0.02 for BDNF, P = 0.0007 for EZH2, respectively, n=3 independent experiments). Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: Subgroups of enriched GO terms shown in have been plotted and visualized using the Metascape ( http://metascape.org ) and Cytoscape5. The program constructs GO term network among terms with a similarity > 0.3, which are connected by edges. Each node represents one enriched GO term, where node size is the number of genes within a GO term. GO terms within the same cluster are visualized with the same color. P -values of the enrichments refer back to the , which shows maximal level of p-value is 1.0E-3. (a) GO term network of downregulated genes in the Nestin-Cre;EZH2 f/f compared with the control. (b) GO term network of downregulated genes in the Neurod6 (Nex)-Cre;EZH2 f/f compared with the control. (c) GO term network of upregulated genes in the Nestin-Cre;EZH2 f/f compared with the control. (d) GO term network of upregulated genes in the Neurod6 (Nex)-Cre;EZH2 f/f compared with the control.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Construct

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Representative western blot images showing the expression of EZH2 and PAK3 in hippocampal neurons after different period of culturing. (b) Representative western blot images showing increased level of PAK3 in neuronal lysis from EZH2 Δ/Δ mice, compared to that from control mice. (c) Diagram showing the PAK3 promoter region, which is artificially separated into three regions, R1 to R3. (d) Representative PCR gel images showing H3K27me3 antibody associated ChIP analysis using P7 mouse cortical tissues. (e) Quantitative reverse transcription PCR analysis of the H3K27me3 ChIP assay shown in (d) (P=0.029, n=3 independent experiments). (f) Representative images of dendritic spines of cultured control neurons, EZH2 Δ/Δ neurons, and EZH2 Δ/Δ neurons expressing dominant negative PAK3 (PAK3 DN ). Note that repression of PAK3 function was able to reduced the increased spine density in EZH2 Δ/Δ neurons. Scale bar, 5µm (g) Quantification of spine densities shown in (f) (n=26, 31, and 30 neurons for the control, EZH2 Δ/Δ and PAK3 DN neurons, respectively, from 4 independent experiments. P =0.0079 between control and EZH2 Δ/Δ neurons; P = 0.0293 between EZH2 Δ/Δ and PAK3 DN neurons). Data are presented as mean ± SEM. n.s., no significant difference, * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Western Blot, Expressing, Lysis, Cell Culture, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Left: diagram of Y maze spontaneous alternation test for examining spatial working memory and spatial recognition memory. Right: diagram of the novel objective recognition test. (b) Quantification of the percentage of correct alternation between Y maze arms. The result revealed that EZH2 Δ/Δ mutant mice have impaired spatial working memory. P =3.02523E-05, n=12 for either EZH2 Δ/Δ or control mice. (c) Quantification of the percentage of time spent in the novel arm relative to the total duration of visits in the three arms during the test phase. Note that EZH2 Δ/Δ mutant mice showed reduced recognition memory. n=25 for control mice and n=18 for EZH2 Δ/Δ mice, P =0.0174. (d) Quantification of novel object recognition memory in EZH2 Δ/Δ mutant mice. Note that EZH2 Δ/Δ mice spent shorter percentage time in novel object. n=15 for control mice and n=20 for EZH2 Δ/Δ mice, P =0.0423. (e) Quantification of the percentage of alternation between Y maze arms. The result revealed that both male and female EZH2 Δ/Δ mutant mice have impaired short-term working memory. Female: n=10 for control mice and n=16 for EZH2 Δ/Δ mice, P =0.01; Male: n=15 for control mice and n=9 for EZH2 Δ/Δ mice, P =0.001. (f) Quantification of the percentage of time spent in novel arms. The result revealed that both male and female EZH2 Δ/Δ mice have impaired short-term working memory. Female: n=10 for control mice and n=16 for EZH2 Δ/Δ mice, P =0.0265; Male: n=15 for control mice and n=9 for EZH2 Δ/Δ mice, P =0.0251. Data are presented as mean ± SEM. n.s., no significant difference, * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, and cognitive behavior of mice

doi: 10.1101/582908

Figure Lengend Snippet: (a) Quantification of time spent in the open arms of the elevated plus maze. Female: n=10 mice for control mice and n=16 for Ezh2 Δ/Δ mice, P =0.6808; male: n=15 for control mice and n=9 for Ezh2 Δ/Δ mice, P =0.6093. (b) Quantification of time spent in the closed arms of the elevated plus maze. Female: n=10 for the control group and n=16 for the Ezh2 Δ/Δ group, P =0.6808; male: n=15 for control mice and n=9 for Ezh2 Δ/Δ mice, P =0.6093. (c-e) Quantification of alterations in time spent peripheral zone (c), central zone (d), and the rearing behavior (e). Female: n=10 for control mice and n=16 for Ezh2 Δ/Δ mice; male: n=15 for control mice and n=9 for Ezh2 Δ/Δ mice. Open field peripheral: P =0.8551 for female mice and P =0.2052 for male mice. Open field center: P =0.8561 for female mice and P =0.3643 for male mice. Open field rearing: P =0.4703 for female mice and P = 0.1854 for male mice. Data are presented as mean ± SEM. n.s., no significant difference, * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: 612666), anti-EZH2 1:1000 (Cell signaling, Cat.no.

Techniques:

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: 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

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