anti ezh2  (Millipore)

 
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    Name:
    Anti EZH2 antibody
    Description:

    Catalog Number:
    E6906
    Price:
    None
    Applications:
    Anti-EZH2 antibody produced in rabbit is suitable for indirect immunofluorescence at a dilution of 1:500-1:1,000 using HEK-293T cells fixed with paraformaldehyde-triton and for western blotting at a dilution of 1:500-1:1,000 using K562 cell lysates.
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    Structured Review

    Millipore anti ezh2
    Anti EZH2 antibody

    https://www.bioz.com/result/anti ezh2/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ezh2 - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE"

    Article Title: Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.007

    HOXA11-AS Can Recruit EZH2 and LSD1 to Silence RND3 Expression (A) Cell fractionation assays indicated that HOXA11-AS is mostly located in the nucleus. GAPDH and U1 acted as markers of the cytoplasm and nucleus, respectively. (B) Bioinformatics were used to predict the possibility of interaction of HOXA11-AS . Predictions with probabilities > 0.5 were considered positive. RPISeq predictions are based on random forest (RF) or support vector machine (SVM). (C) RIP experiments were performed, and the coprecipitated RNA was detected by qRT-PCR. (D) In vitro -transcribed pulldown assays showed that HOXA11-AS could retrieve EZH2 and LSD1 in HTR-8/SVneo cells but not G9a. G9a was a negative control. (E) Western blot assays detected the expression of RND3 after silenced EZH2 after si-RNA transfection in HTR-8/SVneo cells. (F) The enrichment of EZH2 /H3K27me3 and LSD1 /H3K4me2 in the promoter regions of RND3 was identified via ChIP assays, and this enrichment was decreased after HOXA11-AS knockdown in the HTR-8/SVneo cell line. Antibody directed against immunoglobulin G (IgG) was used as a negative control. Values represent the mean ± SEM from three independent experiments. **p
    Figure Legend Snippet: HOXA11-AS Can Recruit EZH2 and LSD1 to Silence RND3 Expression (A) Cell fractionation assays indicated that HOXA11-AS is mostly located in the nucleus. GAPDH and U1 acted as markers of the cytoplasm and nucleus, respectively. (B) Bioinformatics were used to predict the possibility of interaction of HOXA11-AS . Predictions with probabilities > 0.5 were considered positive. RPISeq predictions are based on random forest (RF) or support vector machine (SVM). (C) RIP experiments were performed, and the coprecipitated RNA was detected by qRT-PCR. (D) In vitro -transcribed pulldown assays showed that HOXA11-AS could retrieve EZH2 and LSD1 in HTR-8/SVneo cells but not G9a. G9a was a negative control. (E) Western blot assays detected the expression of RND3 after silenced EZH2 after si-RNA transfection in HTR-8/SVneo cells. (F) The enrichment of EZH2 /H3K27me3 and LSD1 /H3K4me2 in the promoter regions of RND3 was identified via ChIP assays, and this enrichment was decreased after HOXA11-AS knockdown in the HTR-8/SVneo cell line. Antibody directed against immunoglobulin G (IgG) was used as a negative control. Values represent the mean ± SEM from three independent experiments. **p

    Techniques Used: Expressing, Cell Fractionation, Plasmid Preparation, Quantitative RT-PCR, In Vitro, Negative Control, Western Blot, Transfection, Chromatin Immunoprecipitation

    2) Product Images from "Long non‐coding RNA HEIH suppresses the expression of TP53 through enhancer of zeste homolog 2 in oesophageal squamous cell carcinoma. Long non‐coding RNA HEIH suppresses the expression of TP53 through enhancer of zeste homolog 2 in oesophageal squamous cell carcinoma"

    Article Title: Long non‐coding RNA HEIH suppresses the expression of TP53 through enhancer of zeste homolog 2 in oesophageal squamous cell carcinoma. Long non‐coding RNA HEIH suppresses the expression of TP53 through enhancer of zeste homolog 2 in oesophageal squamous cell carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15673

    Validation in Clinical Samples. (A) Expression levels of lncRNA‐HEIH and TP53 were negatively correlated in ESCC samples, as measured by real‐time PCR. The relative expression values (normalized to β‐actin) were subjected to Pearson correlation analysis. (B) ChIP analyses of ESCC and adjacent tissues (n = 12) were conducted on the lncRNA‐HEIH promoter regions using anti‐EZH2 and H3K27me3. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent experiments
    Figure Legend Snippet: Validation in Clinical Samples. (A) Expression levels of lncRNA‐HEIH and TP53 were negatively correlated in ESCC samples, as measured by real‐time PCR. The relative expression values (normalized to β‐actin) were subjected to Pearson correlation analysis. (B) ChIP analyses of ESCC and adjacent tissues (n = 12) were conducted on the lncRNA‐HEIH promoter regions using anti‐EZH2 and H3K27me3. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent experiments

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    lncRNA‐HEIH‐induced down‐expression of TP53 requires EZH2. (A) RNA immunoprecipitation (RIP) experiments were performed using the EZH2 antibody to immunoprecipitate (IP) and a primer to detect lncRNA‐HEIH. (B) RIP enrichment was determined as lncRNA‐HEIH associated with EZH2 IP relative to an input control. (C‐F) ChIP analyses of lncRNA‐HEIH‐overexpressing Eca109 cells (Overexpression‐1 and Overexpression‐2) were conducted on TP53 (primer set a and b) and GAPDH (primer c and d) promoter regions using the indicated antibodies. Enrichment was determined relative to input controls. Asterisk indicates a significant change ( P
    Figure Legend Snippet: lncRNA‐HEIH‐induced down‐expression of TP53 requires EZH2. (A) RNA immunoprecipitation (RIP) experiments were performed using the EZH2 antibody to immunoprecipitate (IP) and a primer to detect lncRNA‐HEIH. (B) RIP enrichment was determined as lncRNA‐HEIH associated with EZH2 IP relative to an input control. (C‐F) ChIP analyses of lncRNA‐HEIH‐overexpressing Eca109 cells (Overexpression‐1 and Overexpression‐2) were conducted on TP53 (primer set a and b) and GAPDH (primer c and d) promoter regions using the indicated antibodies. Enrichment was determined relative to input controls. Asterisk indicates a significant change ( P

    Techniques Used: Expressing, Immunoprecipitation, Chromatin Immunoprecipitation, Over Expression

    3) Product Images from "Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis"

    Article Title: Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2020.12.008

    CLU transcriptional activation by JMJD3 correlates with methylation (A) Western blot analysis and qRT-PCR of the effect of FOXO6 knockdown on JMJD3 protein (normalized to β-actin) and mRNA levels, respectively, in EC cells. (B) qRT-PCR detection of the effect of FOXO6 knockdown on CLU mRNA level in EC cells. (C) ChIP-qPCR detection of H3K27me3 enrichment in CLU promoter region after FOXO6 knockdown in Kyse30 cells. (D) ChIP-qPCR detection of JMJD3 enrichment in CLU promoter region after FOXO6 knockdown in Kyse30 cells. (E) The enrichment of JMJD3 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. (F) The enrichment of H3K27me3 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. (G) The enrichment of JMJD3 EZH2 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. Measurement data were expressed as mean ± standard deviation and compared by independent sample t test between two groups and by one-way ANOVA among multiple groups. The experiment was repeated three times independently. ∗p
    Figure Legend Snippet: CLU transcriptional activation by JMJD3 correlates with methylation (A) Western blot analysis and qRT-PCR of the effect of FOXO6 knockdown on JMJD3 protein (normalized to β-actin) and mRNA levels, respectively, in EC cells. (B) qRT-PCR detection of the effect of FOXO6 knockdown on CLU mRNA level in EC cells. (C) ChIP-qPCR detection of H3K27me3 enrichment in CLU promoter region after FOXO6 knockdown in Kyse30 cells. (D) ChIP-qPCR detection of JMJD3 enrichment in CLU promoter region after FOXO6 knockdown in Kyse30 cells. (E) The enrichment of JMJD3 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. (F) The enrichment of H3K27me3 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. (G) The enrichment of JMJD3 EZH2 at the promoter region of CLU was detected by ChIP-qPCR in response to overexpression of JMJD3 in Kyse30 cells. Measurement data were expressed as mean ± standard deviation and compared by independent sample t test between two groups and by one-way ANOVA among multiple groups. The experiment was repeated three times independently. ∗p

    Techniques Used: Activation Assay, Methylation, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Over Expression, Standard Deviation

    4) Product Images from "Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity"

    Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

    Journal: eLife

    doi: 10.7554/eLife.38080

    Meg3 facilitates the non-stoichiometric interaction of the PRC2 complex and Jarid2 ( A ) Ezh2, Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro -transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value
    Figure Legend Snippet: Meg3 facilitates the non-stoichiometric interaction of the PRC2 complex and Jarid2 ( A ) Ezh2, Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro -transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value

    Techniques Used: Immunoprecipitation, In Vitro

    5) Product Images from "Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression"

    Article Title: Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

    Journal: Epigenetics

    doi: 10.4161/epi.27957

    Figure 1. Expression of MST1 is inversely correlated with those of MYC and EZH2 in PC cells. ( A ) Analysis of Oncomine microarray data for MST1 mRNA expression changes in multiple cancer cell lines. ( B–E) Analysis of MST1 ( B ), MYC ( C ) and
    Figure Legend Snippet: Figure 1. Expression of MST1 is inversely correlated with those of MYC and EZH2 in PC cells. ( A ) Analysis of Oncomine microarray data for MST1 mRNA expression changes in multiple cancer cell lines. ( B–E) Analysis of MST1 ( B ), MYC ( C ) and

    Techniques Used: Expressing, Microarray

    Figure 6. Suppression of MST1 expression causes androgen independence and resistance to MYC or EZH2 inhibitor-induced growth retardation. ( A and B ) Effects of MST1 knockdown on an androgen-independent LNCaP cell growth in vitro. LNCaP cells were
    Figure Legend Snippet: Figure 6. Suppression of MST1 expression causes androgen independence and resistance to MYC or EZH2 inhibitor-induced growth retardation. ( A and B ) Effects of MST1 knockdown on an androgen-independent LNCaP cell growth in vitro. LNCaP cells were

    Techniques Used: Expressing, In Vitro

    Figure 5. EZH2-mediated chromatin remodeling and DNA methylation attenuates MST1 promoter activation and expression. ( A ) MST1 promoter luciferase reporter activity in C4–2 cells after transient transfection with pMST1-Luc2 reporter or mock vector,
    Figure Legend Snippet: Figure 5. EZH2-mediated chromatin remodeling and DNA methylation attenuates MST1 promoter activation and expression. ( A ) MST1 promoter luciferase reporter activity in C4–2 cells after transient transfection with pMST1-Luc2 reporter or mock vector,

    Techniques Used: DNA Methylation Assay, Activation Assay, Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    Figure 4. Attenuation of MST1 expression by MYC through EZH2 and miR-26a/b. ( A and B ) Analysis of MST1, EZH2 and MYC mRNA levels by RT-qPCR in C4–2 cells after treatment with increasing doses (0, 20, 40, 60 µM) of 10058-F4 for 72h and
    Figure Legend Snippet: Figure 4. Attenuation of MST1 expression by MYC through EZH2 and miR-26a/b. ( A and B ) Analysis of MST1, EZH2 and MYC mRNA levels by RT-qPCR in C4–2 cells after treatment with increasing doses (0, 20, 40, 60 µM) of 10058-F4 for 72h and

    Techniques Used: Expressing, Quantitative RT-PCR

    Figure 3. Overexpression of EZH2 negatively regulates MST1 expression in PC cells. ( A and B ). Analysis of MST1 mRNA levels by RT-qPCR ( A ) and MST1, EZH2, and β-Actin protein levels by WB ( B ) in C4–2 cells after treatment with DMSO
    Figure Legend Snippet: Figure 3. Overexpression of EZH2 negatively regulates MST1 expression in PC cells. ( A and B ). Analysis of MST1 mRNA levels by RT-qPCR ( A ) and MST1, EZH2, and β-Actin protein levels by WB ( B ) in C4–2 cells after treatment with DMSO

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Western Blot

    6) Product Images from "Long intergenic non-coding RNA 00152 promotes renal cell carcinoma progression by epigenetically suppressing P16 and negatively regulates miR-205"

    Article Title: Long intergenic non-coding RNA 00152 promotes renal cell carcinoma progression by epigenetically suppressing P16 and negatively regulates miR-205

    Journal: American Journal of Cancer Research

    doi:

    LINC00152 was negatively correlated with miR-205 in RCC. A: Chromatin immunoprecipitation-qPCR analysis of EZH2 and LSD1 occupancy and H3K27me3 binding to the P16 promoter regions in ACHN and 786-O cells, and IgG as a negative control. Results are represented
    Figure Legend Snippet: LINC00152 was negatively correlated with miR-205 in RCC. A: Chromatin immunoprecipitation-qPCR analysis of EZH2 and LSD1 occupancy and H3K27me3 binding to the P16 promoter regions in ACHN and 786-O cells, and IgG as a negative control. Results are represented

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control

    LINC00152 represses P16 by binding with EZH2, LSD1 and H3k27me3 in RCC cells. A: Subcellular localization of LINC00152 was determined using fractionation. After nuclear and cytoplasm separation in ACHN and 786-O cells, RNA was extracted from both fractions
    Figure Legend Snippet: LINC00152 represses P16 by binding with EZH2, LSD1 and H3k27me3 in RCC cells. A: Subcellular localization of LINC00152 was determined using fractionation. After nuclear and cytoplasm separation in ACHN and 786-O cells, RNA was extracted from both fractions

    Techniques Used: Binding Assay, Fractionation

    7) Product Images from "MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines *"

    Article Title: MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.750950

    Knockdown of MEG3 affected the TGF-β-induced regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with retroviruses expressing control shRNA or MEG3 shRNA#1 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (*, p
    Figure Legend Snippet: Knockdown of MEG3 affected the TGF-β-induced regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with retroviruses expressing control shRNA or MEG3 shRNA#1 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (*, p

    Techniques Used: Methylation, Infection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Knockdown of JARID2 inhibited MEG3 -induced changes in the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p
    Figure Legend Snippet: Knockdown of JARID2 inhibited MEG3 -induced changes in the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p

    Techniques Used: Methylation, Infection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Overexpression of MEG3 affected the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p
    Figure Legend Snippet: Overexpression of MEG3 affected the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p

    Techniques Used: Over Expression, Methylation, Infection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    MEG3 interacted with JARID2 and stimulated the interaction and recruitment of JARID2 and EZH2 on the regulatory regions of CDH1 , miR-200a, and miR-200c genes for histone H3K27 methylation in A549 cells. A, interaction of MEG3 and JARID2 detected by RIP. A549 cells were infected with the various combinations (as indicated) of the retroviruses expressing MEG3 and FLAG-tagged JARID2. The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG) or anti-FLAG antibody, and the co-precipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 in the precipitates. n.d. means not detected. B–E, A549 cells were infected with the various combinations (as indicated) of the retroviruses without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and FLAG-tagged JARID2 on the regulatory regions of CDH1 ( B ), miR-200b/200a/429 ( C ), miR-200c/141 ( D ), and GAPDH genes ( E ) are shown (*, p
    Figure Legend Snippet: MEG3 interacted with JARID2 and stimulated the interaction and recruitment of JARID2 and EZH2 on the regulatory regions of CDH1 , miR-200a, and miR-200c genes for histone H3K27 methylation in A549 cells. A, interaction of MEG3 and JARID2 detected by RIP. A549 cells were infected with the various combinations (as indicated) of the retroviruses expressing MEG3 and FLAG-tagged JARID2. The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG) or anti-FLAG antibody, and the co-precipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 in the precipitates. n.d. means not detected. B–E, A549 cells were infected with the various combinations (as indicated) of the retroviruses without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and FLAG-tagged JARID2 on the regulatory regions of CDH1 ( B ), miR-200b/200a/429 ( C ), miR-200c/141 ( D ), and GAPDH genes ( E ) are shown (*, p

    Techniques Used: Methylation, Infection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    8) Product Images from "Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency"

    Article Title: Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency

    Journal: mBio

    doi: 10.1128/mBio.00133-17

    Distribution of histone methyltransferases and methylated histones on HIV proviruses following CRISPR disruption. (A) EZH2 and EHMT2 disruption. (B) JARID2 disruption. ChIP assays were performed with E4 cells infected with the CRISPRs indicated by using primers to the HIV LTR and downstream regions ( 27 ). HIV DNA levels were calculated as percentages of the input. Error bars represent the SEM of three separate real-time PCR measurements.
    Figure Legend Snippet: Distribution of histone methyltransferases and methylated histones on HIV proviruses following CRISPR disruption. (A) EZH2 and EHMT2 disruption. (B) JARID2 disruption. ChIP assays were performed with E4 cells infected with the CRISPRs indicated by using primers to the HIV LTR and downstream regions ( 27 ). HIV DNA levels were calculated as percentages of the input. Error bars represent the SEM of three separate real-time PCR measurements.

    Techniques Used: Methylation, CRISPR, Chromatin Immunoprecipitation, Infection, Real-time Polymerase Chain Reaction

    PRC2 is required for silent integration of HIV-1 in Jurkat T cells. (A) Experimental scheme. (B) Representative flow data measuring levels of H3K27me3 and HIV infection in cells expressing EZH2 or scrambled shRNA in untreated cells. (C) Cells treated with 1 µM SAHA. Black, cell counts per quadrant; red, distribution of GFP + cells in high-H3K27me3 gate; blue, distribution of GFP + cells in low-H3K27me3 gate. (D) Relative silencing of HIV-1 from cells treated with shRNAs targeting different subunits of PRC2. (Top) Inducible virus in high- and low-H3K27me3 populations. (Bottom) Relative silencing after knockdown of each of the PRC2 subunits. Error bars represent the SEM of three separate experiments.
    Figure Legend Snippet: PRC2 is required for silent integration of HIV-1 in Jurkat T cells. (A) Experimental scheme. (B) Representative flow data measuring levels of H3K27me3 and HIV infection in cells expressing EZH2 or scrambled shRNA in untreated cells. (C) Cells treated with 1 µM SAHA. Black, cell counts per quadrant; red, distribution of GFP + cells in high-H3K27me3 gate; blue, distribution of GFP + cells in low-H3K27me3 gate. (D) Relative silencing of HIV-1 from cells treated with shRNAs targeting different subunits of PRC2. (Top) Inducible virus in high- and low-H3K27me3 populations. (Bottom) Relative silencing after knockdown of each of the PRC2 subunits. Error bars represent the SEM of three separate experiments.

    Techniques Used: Flow Cytometry, Infection, Expressing, shRNA

    EZH2 and EHMT2 are required for the establishment of latent HIV-1 in primary Th17 cells. (A) Experimental design. (B) Reactivation of latent proviruses by Dynabeads Human T-Activator CD3/CD28, IL-15 (50 ng/ml), or ConA (5 µg/ml) in cells pretreated with DMSO, 100 nM GSK-343, EPZ-6438, or UNC-0638.
    Figure Legend Snippet: EZH2 and EHMT2 are required for the establishment of latent HIV-1 in primary Th17 cells. (A) Experimental design. (B) Reactivation of latent proviruses by Dynabeads Human T-Activator CD3/CD28, IL-15 (50 ng/ml), or ConA (5 µg/ml) in cells pretreated with DMSO, 100 nM GSK-343, EPZ-6438, or UNC-0638.

    Techniques Used:

    CRISPR-mediated depletion of EZH2 or JARID2 reactivates latent HIV-1 in E4 cells. Panels: A, EZH2-disrupted cells; B, JARID2-disrupted cells; C, EHMT2 (G9a)-disrupted cells. (Top) Western blot assays showing histone methyltransferases and methylated histones. (Bottom) Quantification of HIV-1 reactivation. E4 cells were infected with CRISPR-Cas9-expressing viruses targeting the proteins indicated. Reactivation of HIV-1 by stimulation with SAHA (1 μM) overnight was measured 7 days postinfection by FACS analysis. Error bars represent the SEM of three separate experiments.
    Figure Legend Snippet: CRISPR-mediated depletion of EZH2 or JARID2 reactivates latent HIV-1 in E4 cells. Panels: A, EZH2-disrupted cells; B, JARID2-disrupted cells; C, EHMT2 (G9a)-disrupted cells. (Top) Western blot assays showing histone methyltransferases and methylated histones. (Bottom) Quantification of HIV-1 reactivation. E4 cells were infected with CRISPR-Cas9-expressing viruses targeting the proteins indicated. Reactivation of HIV-1 by stimulation with SAHA (1 μM) overnight was measured 7 days postinfection by FACS analysis. Error bars represent the SEM of three separate experiments.

    Techniques Used: CRISPR, Western Blot, Methylation, Infection, Expressing, FACS

    9) Product Images from "Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression"

    Article Title: Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1600070113

    Snapshots of ChIP-seq data from ENCODE/Broad Institute for the K562 cell line. UCSC genome browser visualization of ITLN2 ( A ), SLC4A1 ( B ), and KLHDC8A ( C ) gene loci and ENCODE/Broad Institute ChIP-seq data using H3K4me3, EZH2, and H3K27me3 antibodies
    Figure Legend Snippet: Snapshots of ChIP-seq data from ENCODE/Broad Institute for the K562 cell line. UCSC genome browser visualization of ITLN2 ( A ), SLC4A1 ( B ), and KLHDC8A ( C ) gene loci and ENCODE/Broad Institute ChIP-seq data using H3K4me3, EZH2, and H3K27me3 antibodies

    Techniques Used: Chromatin Immunoprecipitation

    10) Product Images from "LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells"

    Article Title: LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171070

    DANCR epigenetically suppresses lncRNA-LET expression through association with EZH2 and HDAC3 ( A ) The DANCR-overexpressed AGS cells were treated with 5 μM DZNep and/or 1 μM SAHA for 48 h, and the relative expression of lncRNA-LET was detected by qPCR. ( B ) The DANCR-overexpressed AGS cells were transfected with EZH2 and/or HDAC3. After 48 h, the relative expression of lncRNA-LET was detected by qPCR. ( C ) DANCR RNA levels in immunoprecipitates by EZH2 or HDAC3 were determined by qPCR. DANCR RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. ( D ) EZH2 and HDAC3 protein levels in immunoprecipitates with biotin-labeled DANCR RNA were evaluated by Western blot. ( E ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-silenced BCG-823 cells. ( F ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-overexpressed AGS cells. ( G ) BCG-823 cells were transfected with DANCR siRNAs for 48 h. After immunoprecipitating endogenous EZH2, bound HDAC3 was subjected to Western blotting. All experiments were repeated three times. Data are shown as mean ± SD; * P
    Figure Legend Snippet: DANCR epigenetically suppresses lncRNA-LET expression through association with EZH2 and HDAC3 ( A ) The DANCR-overexpressed AGS cells were treated with 5 μM DZNep and/or 1 μM SAHA for 48 h, and the relative expression of lncRNA-LET was detected by qPCR. ( B ) The DANCR-overexpressed AGS cells were transfected with EZH2 and/or HDAC3. After 48 h, the relative expression of lncRNA-LET was detected by qPCR. ( C ) DANCR RNA levels in immunoprecipitates by EZH2 or HDAC3 were determined by qPCR. DANCR RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. ( D ) EZH2 and HDAC3 protein levels in immunoprecipitates with biotin-labeled DANCR RNA were evaluated by Western blot. ( E ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-silenced BCG-823 cells. ( F ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-overexpressed AGS cells. ( G ) BCG-823 cells were transfected with DANCR siRNAs for 48 h. After immunoprecipitating endogenous EZH2, bound HDAC3 was subjected to Western blotting. All experiments were repeated three times. Data are shown as mean ± SD; * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, RNA Expression, Labeling, Western Blot, Chromatin Immunoprecipitation

    11) Product Images from "MicroRNA-365 targets multiple oncogenes to inhibit proliferation, invasion, and self-renewal of aggressive endometrial cancer cells"

    Article Title: MicroRNA-365 targets multiple oncogenes to inhibit proliferation, invasion, and self-renewal of aggressive endometrial cancer cells

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S174889

    Decrease in expression of EZH2 and FOS suppresses invasion, proliferation and CSC-like phenotypes of cancerous endometrial cancer cells. Notes: The results indicate the number of cells that underwent apoptosis, sphere formation assay, cells senescence, and invasion fold change in ( A ) SPAC-1-L and ( B ) HEC-50 cells after 72 hours of transfection. ** P
    Figure Legend Snippet: Decrease in expression of EZH2 and FOS suppresses invasion, proliferation and CSC-like phenotypes of cancerous endometrial cancer cells. Notes: The results indicate the number of cells that underwent apoptosis, sphere formation assay, cells senescence, and invasion fold change in ( A ) SPAC-1-L and ( B ) HEC-50 cells after 72 hours of transfection. ** P

    Techniques Used: Expressing, Tube Formation Assay, Transfection

    EZH2 and FOS are direct targets of miR-365. Notes: ( A ) Overexpression of miR-365 resulted in suppression of FLRT3, NEK7, and UBE2D1 in SPAC-1-L cells, whereas suppression of miR-365 resulted in upregulation of these three genes in HOUA-I cells. ( B) Overexpression of miR-365 resulted in suppression of EZH2 and FOS in SPAC-1-L cells, whereas suppression of miR-365 caused upregulation of EZH2 and FOS in HOUA-I cells. ( C) Results of Western blot studies after transfection of SPAC-1-L cells with miR-365 on the levels of EZH2 and FOS or with AS-365 in HOUA-I cells. Abbreviations: ANC, anti-miRNA negative control; NC, negative control.
    Figure Legend Snippet: EZH2 and FOS are direct targets of miR-365. Notes: ( A ) Overexpression of miR-365 resulted in suppression of FLRT3, NEK7, and UBE2D1 in SPAC-1-L cells, whereas suppression of miR-365 resulted in upregulation of these three genes in HOUA-I cells. ( B) Overexpression of miR-365 resulted in suppression of EZH2 and FOS in SPAC-1-L cells, whereas suppression of miR-365 caused upregulation of EZH2 and FOS in HOUA-I cells. ( C) Results of Western blot studies after transfection of SPAC-1-L cells with miR-365 on the levels of EZH2 and FOS or with AS-365 in HOUA-I cells. Abbreviations: ANC, anti-miRNA negative control; NC, negative control.

    Techniques Used: Over Expression, Western Blot, Transfection, Negative Control

    An inverse correlation existed between down-regulation of miR-365 and up-regulation of EZH2 and FOS in endometrial cancer tissue. Notes: ( A ) Results of qRT-PCR showed the suppression of miR-365 in 12 pairs of selected cancerous EC tissues (CT) and the normal tissues (NT) adjacent to them. ( B–D ) The qRT-PCR results of EZH2 and FOS in eight pairs of EC, along with those of adjacent normal tissues. ( E ) Images after IHC analysis of EZH2 and FOS in tissues of patients suggested elevated expression of EZH2 and FOS in cancer cells and in cells reported to be benign. ** P
    Figure Legend Snippet: An inverse correlation existed between down-regulation of miR-365 and up-regulation of EZH2 and FOS in endometrial cancer tissue. Notes: ( A ) Results of qRT-PCR showed the suppression of miR-365 in 12 pairs of selected cancerous EC tissues (CT) and the normal tissues (NT) adjacent to them. ( B–D ) The qRT-PCR results of EZH2 and FOS in eight pairs of EC, along with those of adjacent normal tissues. ( E ) Images after IHC analysis of EZH2 and FOS in tissues of patients suggested elevated expression of EZH2 and FOS in cancer cells and in cells reported to be benign. ** P

    Techniques Used: Quantitative RT-PCR, Immunohistochemistry, Expressing

    Western blot analysis of EZH2 and FOS in EC cells. Note: ( A , B ) Results of Western blot analysis of EZH2 and FOS in SPAC-1-L and HEC-50 cells after transfection with specific siRNAs (siEZH2 and siFOS) or control siRNA (siNC). Abbreviation: NC, negative control.
    Figure Legend Snippet: Western blot analysis of EZH2 and FOS in EC cells. Note: ( A , B ) Results of Western blot analysis of EZH2 and FOS in SPAC-1-L and HEC-50 cells after transfection with specific siRNAs (siEZH2 and siFOS) or control siRNA (siNC). Abbreviation: NC, negative control.

    Techniques Used: Western Blot, Transfection, Negative Control

    Luciferase study confirmed EZH2 and FOS as direct targets of miR-365. Notes: The luciferase reporter assays identified both EZH2 and FOS as direct targets of miR-365 in both ( A ) SPAC-1-L and ( B) HOUA-I cells. ** P
    Figure Legend Snippet: Luciferase study confirmed EZH2 and FOS as direct targets of miR-365. Notes: The luciferase reporter assays identified both EZH2 and FOS as direct targets of miR-365 in both ( A ) SPAC-1-L and ( B) HOUA-I cells. ** P

    Techniques Used: Luciferase

    12) Product Images from "The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation"

    Article Title: The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation

    Journal: Cell Cycle

    doi: 10.4161/cc.22025

    Figure 1. RD cells fail to complete the differentiation program and show high levels of EZH2. Myoblasts (C2C12) and RMS cells (RD) were grown to 90% and induced to differentiate by serum withdrawal and addition of 2% horse serum (DM) for 96 h.
    Figure Legend Snippet: Figure 1. RD cells fail to complete the differentiation program and show high levels of EZH2. Myoblasts (C2C12) and RMS cells (RD) were grown to 90% and induced to differentiate by serum withdrawal and addition of 2% horse serum (DM) for 96 h.

    Techniques Used:

    Figure 6. EZH2 ablation restores the phosphorylation of RNA polymerase II on muscle-specific genes promoting transcriptional activation. Silencing of EZH2 promotes the recruitment of transcriptional activation complex at MyoD-dependent genes promoting
    Figure Legend Snippet: Figure 6. EZH2 ablation restores the phosphorylation of RNA polymerase II on muscle-specific genes promoting transcriptional activation. Silencing of EZH2 promotes the recruitment of transcriptional activation complex at MyoD-dependent genes promoting

    Techniques Used: Activation Assay

    Figure 5. EZH2 ablation restores MyoD binding on muscle-specific genes. ChIP assays, performed in RD wild type and EZH2 knockdown 72 h following differentiation induction, show (A) the recovery of MyoD binding at myogenin and Myh regulatory regions
    Figure Legend Snippet: Figure 5. EZH2 ablation restores MyoD binding on muscle-specific genes. ChIP assays, performed in RD wild type and EZH2 knockdown 72 h following differentiation induction, show (A) the recovery of MyoD binding at myogenin and Myh regulatory regions

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    Figure 4. EZH2 inhibits the expression of skeletal muscle-specific genes. Differentiation induction in absence of EZH2 results in a significant increase of muscle-specific genes. (A) Real-time qPCR was performed using cDNA from RD wild type and
    Figure Legend Snippet: Figure 4. EZH2 inhibits the expression of skeletal muscle-specific genes. Differentiation induction in absence of EZH2 results in a significant increase of muscle-specific genes. (A) Real-time qPCR was performed using cDNA from RD wild type and

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Figure 2. EZH2 binds muscle-specific gene promoters in RD cells. Chromatin immunoprecipitation revealed that EZH2 binds muscle-specific gene promoters in RD cells. The chromatin-immunoprecipitated DNA was amplified by real-time PCR using specific
    Figure Legend Snippet: Figure 2. EZH2 binds muscle-specific gene promoters in RD cells. Chromatin immunoprecipitation revealed that EZH2 binds muscle-specific gene promoters in RD cells. The chromatin-immunoprecipitated DNA was amplified by real-time PCR using specific

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    Figure 3. EZH2-knockdown RD cells show partial reversion of tumor phenotype. Real-time PCR (A) and immunoblotting (B) show a decrease of EZH2 levels of 80% in EZH2 knockdown RD (kd) compared with RD wild type (wt) and scramble (scr). (C) RD wild
    Figure Legend Snippet: Figure 3. EZH2-knockdown RD cells show partial reversion of tumor phenotype. Real-time PCR (A) and immunoblotting (B) show a decrease of EZH2 levels of 80% in EZH2 knockdown RD (kd) compared with RD wild type (wt) and scramble (scr). (C) RD wild

    Techniques Used: Real-time Polymerase Chain Reaction

    13) Product Images from "Long non-coding RNA SNHG15 inhibits P15 and KLF2 expression to promote pancreatic cancer proliferation through EZH2-mediated H3K27me3"

    Article Title: Long non-coding RNA SNHG15 inhibits P15 and KLF2 expression to promote pancreatic cancer proliferation through EZH2-mediated H3K27me3

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20359

    SNHG15 epigenetically silences P15 and KLF2 transcription by binding to EZH2 (A) qRT-PCR analysis of SNHG15 nuclear and cytoplasmic expression levels in AsPC-1 and BxPC-3 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. (B) RIP experiments were performed in AsPC-1 and BxPC-3 cells, and the coprecipitated RNA was subjected to qRT-PCR for SNHG15. The fold enrichment of SNHG15 in EZH2/SUZ12 RIP is relative to its matched IgG control. (C) The levels of p15, p16, p21, p27, p57, KLF2 and PTEN mRNA were determined by qRT–PCR when knockdown of SNHG15. (D) The p15 and KLF2 protein levels were determined by western blot in SNHG15 knockdown in AsPC-1 and BxPC-3 cells. (E) The p15 and KLF2 expression levels were determined by qRT-PCR in AsPC-1 and BxPC-3 cells transfected with si-EZH2 1# or 2#. (F) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the p15 and KLF2 promoters in AsPC-1 and BxPC-3 cells treated with si-SNHG15 3# (48 h) or si-NC; IgG as a negative control. Error bars indicate mean ± standard errors of the mean. * P
    Figure Legend Snippet: SNHG15 epigenetically silences P15 and KLF2 transcription by binding to EZH2 (A) qRT-PCR analysis of SNHG15 nuclear and cytoplasmic expression levels in AsPC-1 and BxPC-3 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. (B) RIP experiments were performed in AsPC-1 and BxPC-3 cells, and the coprecipitated RNA was subjected to qRT-PCR for SNHG15. The fold enrichment of SNHG15 in EZH2/SUZ12 RIP is relative to its matched IgG control. (C) The levels of p15, p16, p21, p27, p57, KLF2 and PTEN mRNA were determined by qRT–PCR when knockdown of SNHG15. (D) The p15 and KLF2 protein levels were determined by western blot in SNHG15 knockdown in AsPC-1 and BxPC-3 cells. (E) The p15 and KLF2 expression levels were determined by qRT-PCR in AsPC-1 and BxPC-3 cells transfected with si-EZH2 1# or 2#. (F) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the p15 and KLF2 promoters in AsPC-1 and BxPC-3 cells treated with si-SNHG15 3# (48 h) or si-NC; IgG as a negative control. Error bars indicate mean ± standard errors of the mean. * P

    Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing, Marker, Western Blot, Transfection, Chromatin Immunoprecipitation, Negative Control

    14) Product Images from "Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes ▿"

    Article Title: Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00160-11

    Increased expression of EZH2 in differentiating E6/E7 cells. (A) QRT-PCR of EZH2 in differentiated pBabe and E6/E7 cells following CaCl 2 -induced differentiation. Data represent transcripts at 48 h of differentiation for three independent experiments. (B) Using immunohistochemistry detection, the EZH2 protein was shown to be confined to the nucleus in the basal cells of HFK rafts, while more abundant and stronger nuclear staining was observed throughout the epithelial layers of E6/E7 raft sections. Higher levels of P-EZH2-Ser21 were observed in the nuclei of E6/E7 cells than in normal HFKs. Control sections of pBabe and E6/E7 raft sections showed negative nuclei and minimal background staining following incubation of the P-EZH2-Ser21 antibody with a 3-fold excess of blocking peptide (EZH2-BP). (C) Quantification by two observers of the median number of cells positive for EZH2 compared to Ki67 in 10 independent frames from raft sections indicated that EZH2 was expressed at a higher level in E6/E7 cells than in normal HFKs. (D) Western blot showing that E6/E7 cells contain high levels of active Akt compared to pBabe cells. (E) siRNA knockdown of Akt, measured by protein levels and reduced phosphorylation of the Akt substrate GSK-3β, results in a reduced level of P-EZH2-Ser21. (F) Pharmacological reduction of P-Akt results in reduced levels of P-Akt in E6/E7 cells and in a significant increase in H3K27me23 levels in E6/E7 cells compared to pBabe controls.
    Figure Legend Snippet: Increased expression of EZH2 in differentiating E6/E7 cells. (A) QRT-PCR of EZH2 in differentiated pBabe and E6/E7 cells following CaCl 2 -induced differentiation. Data represent transcripts at 48 h of differentiation for three independent experiments. (B) Using immunohistochemistry detection, the EZH2 protein was shown to be confined to the nucleus in the basal cells of HFK rafts, while more abundant and stronger nuclear staining was observed throughout the epithelial layers of E6/E7 raft sections. Higher levels of P-EZH2-Ser21 were observed in the nuclei of E6/E7 cells than in normal HFKs. Control sections of pBabe and E6/E7 raft sections showed negative nuclei and minimal background staining following incubation of the P-EZH2-Ser21 antibody with a 3-fold excess of blocking peptide (EZH2-BP). (C) Quantification by two observers of the median number of cells positive for EZH2 compared to Ki67 in 10 independent frames from raft sections indicated that EZH2 was expressed at a higher level in E6/E7 cells than in normal HFKs. (D) Western blot showing that E6/E7 cells contain high levels of active Akt compared to pBabe cells. (E) siRNA knockdown of Akt, measured by protein levels and reduced phosphorylation of the Akt substrate GSK-3β, results in a reduced level of P-EZH2-Ser21. (F) Pharmacological reduction of P-Akt results in reduced levels of P-Akt in E6/E7 cells and in a significant increase in H3K27me23 levels in E6/E7 cells compared to pBabe controls.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Incubation, Blocking Assay, Western Blot

    Upregulation of EZH2 and loss of H3K27me3 in HSILs. Immunofluorescence costaining of EZH2 and H3K27me3 in HSILs and normal biopsy specimens. Green, H3K27me3; red, EZH2; merge, DAPI plus H3K27me3. High levels of EZH2 were shown to correlate with reduced H3K27me3 staining in HSILs, while the reverse was observed in normal biopsy specimens. While EZH2 was located predominantly in the nucleus in the basal cells of normal tissue, more abundant and stronger nuclear staining was observed throughout epithelial layers of HSILs. The figure is representative of three independent sets of matched HSILs and normal biopsy specimens.
    Figure Legend Snippet: Upregulation of EZH2 and loss of H3K27me3 in HSILs. Immunofluorescence costaining of EZH2 and H3K27me3 in HSILs and normal biopsy specimens. Green, H3K27me3; red, EZH2; merge, DAPI plus H3K27me3. High levels of EZH2 were shown to correlate with reduced H3K27me3 staining in HSILs, while the reverse was observed in normal biopsy specimens. While EZH2 was located predominantly in the nucleus in the basal cells of normal tissue, more abundant and stronger nuclear staining was observed throughout epithelial layers of HSILs. The figure is representative of three independent sets of matched HSILs and normal biopsy specimens.

    Techniques Used: Immunofluorescence, Staining

    Increased expression of EZH2 in E6/E7 cells compared to control pBabe cells. (A) Using QRT-PCR, cycling E6/E7 cells were shown to contain higher levels of EZH2 mRNA than pBabe cells. (B) E6/E7 cells contain increased levels of EZH2 protein compared to pBabe cells. The two protein isoforms, which differ by 44 amino acids, were resolved by 7.5% PAGE. (C) E6/E7 cells contain reduced levels of pRb and p53 as a result of HPV E7- and E6-mediated inactivation, respectively. No effect on SUV39h1 methylase or PCAF acetylase was observed in E6/E7 cells. Data are representative of three independent experiments using cells between passages 5 and 8.
    Figure Legend Snippet: Increased expression of EZH2 in E6/E7 cells compared to control pBabe cells. (A) Using QRT-PCR, cycling E6/E7 cells were shown to contain higher levels of EZH2 mRNA than pBabe cells. (B) E6/E7 cells contain increased levels of EZH2 protein compared to pBabe cells. The two protein isoforms, which differ by 44 amino acids, were resolved by 7.5% PAGE. (C) E6/E7 cells contain reduced levels of pRb and p53 as a result of HPV E7- and E6-mediated inactivation, respectively. No effect on SUV39h1 methylase or PCAF acetylase was observed in E6/E7 cells. Data are representative of three independent experiments using cells between passages 5 and 8.

    Techniques Used: Expressing, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis

    Quantitative changes in polycomb proteins and demethylases in E6/E7 cells and effect of H3K27me3 loss on HOX expression. (A) Western blot analysis of PRC2 core proteins and BMI1 (PRC1) in 60-μg total cell lysates from E6/E7 cells and pBabe cells. The blots show increased expression of EZH2 and downregulation of BMI1 but no significant change in SUZ12 or EED isoform levels in E6/E7 cells compared to pBabe cells. Increased expression of KDM6A was also observed in E6/E7 cells, while KDM6B protein levels remained very low in both cell lines. (B) QRT-PCR analysis of H3K27me3-targeted HOX gene expression in cycling E6/E7 cells compared to pBabe cells. Expression data are presented as C T values normalized to β2-M, and fold changes, calculated using the 2 −ΔΔ C T method, are indicated at the bottom. Data represent three independent experiments. *, P
    Figure Legend Snippet: Quantitative changes in polycomb proteins and demethylases in E6/E7 cells and effect of H3K27me3 loss on HOX expression. (A) Western blot analysis of PRC2 core proteins and BMI1 (PRC1) in 60-μg total cell lysates from E6/E7 cells and pBabe cells. The blots show increased expression of EZH2 and downregulation of BMI1 but no significant change in SUZ12 or EED isoform levels in E6/E7 cells compared to pBabe cells. Increased expression of KDM6A was also observed in E6/E7 cells, while KDM6B protein levels remained very low in both cell lines. (B) QRT-PCR analysis of H3K27me3-targeted HOX gene expression in cycling E6/E7 cells compared to pBabe cells. Expression data are presented as C T values normalized to β2-M, and fold changes, calculated using the 2 −ΔΔ C T method, are indicated at the bottom. Data represent three independent experiments. *, P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

    Global H3K27me3 is reduced and relocated in E6/E7 cells containing increased EZH2 protein. (A) Western blot of nuclear fractions showing reduced H3K27me3 protein in E6/E7 cells compared to pBabe cells. Total histone 3 (H3) and the nuclear loading control TBP are also shown, and the blot is representative of three independent experiments. (B) Immunofluorescence costaining of H3K27me3 and EZH2 in cycling normal HFKs (pBabe) and E6/E7 cells. The loss of H3K27me3 levels was observed in E6/E7 cells containing increased levels of EZH2. Blue, DAPI; red, EZH2; green, H3K27me3. (C) H3K27me3 staining and/or fluorescence was also evaluated using confocal microscopy. In E6/E7 cells containing increased EZH2 protein, reduced H3K27me3 fluorescence was observed compared to that in pBabe cells. Intense H3K27me3-enriched foci were observed in the nuclei of pBabe cells, which localized to pericentric heterochromatin. In contrast, H3K27me3 staining appeared to be diffuse in E6/E7 cells. Blue, DAPI; green, H3K27me3.
    Figure Legend Snippet: Global H3K27me3 is reduced and relocated in E6/E7 cells containing increased EZH2 protein. (A) Western blot of nuclear fractions showing reduced H3K27me3 protein in E6/E7 cells compared to pBabe cells. Total histone 3 (H3) and the nuclear loading control TBP are also shown, and the blot is representative of three independent experiments. (B) Immunofluorescence costaining of H3K27me3 and EZH2 in cycling normal HFKs (pBabe) and E6/E7 cells. The loss of H3K27me3 levels was observed in E6/E7 cells containing increased levels of EZH2. Blue, DAPI; red, EZH2; green, H3K27me3. (C) H3K27me3 staining and/or fluorescence was also evaluated using confocal microscopy. In E6/E7 cells containing increased EZH2 protein, reduced H3K27me3 fluorescence was observed compared to that in pBabe cells. Intense H3K27me3-enriched foci were observed in the nuclei of pBabe cells, which localized to pericentric heterochromatin. In contrast, H3K27me3 staining appeared to be diffuse in E6/E7 cells. Blue, DAPI; green, H3K27me3.

    Techniques Used: Western Blot, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy

    15) Product Images from "A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene"

    Article Title: A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv567

    Model showing PRDM2 as a master regulator of quiescence—regulation of CCNA2 poising in reversible arrest via PRC2 dependent bivalent domain. ( A ) PRDM2 choreographs a genome-wide program to keep quiescent cells in a poised state by repressing myogenic networks to prevent differentiation (left), inducing/maintaining myogenic specification factors (right) and preserving reversibility of the cell cycle program via control of balanced methylation at a newly defined element in CCNA2 intron (center). Knockdown of PRDM2 subverts the quiescence program towards differentiation-coupled irreversible arrest. ( B ) PRDM2 prevents PRC2-mediated silencing of CCNA2 by sequestering EZH2 and preventing H3K27me3 accumulation at the CCNA2 PRE-like element. Thereby, G 0 cells are held in suspended animation, poised to return to active proliferation, with a subsequent option to differentiate if conditions are conducive. PRDM2 knockdown permits EZH2 to accumulate at the PRE, leading to increased H3K27me3, silencing CCNA2 and diverting quiescence toward differentiation.
    Figure Legend Snippet: Model showing PRDM2 as a master regulator of quiescence—regulation of CCNA2 poising in reversible arrest via PRC2 dependent bivalent domain. ( A ) PRDM2 choreographs a genome-wide program to keep quiescent cells in a poised state by repressing myogenic networks to prevent differentiation (left), inducing/maintaining myogenic specification factors (right) and preserving reversibility of the cell cycle program via control of balanced methylation at a newly defined element in CCNA2 intron (center). Knockdown of PRDM2 subverts the quiescence program towards differentiation-coupled irreversible arrest. ( B ) PRDM2 prevents PRC2-mediated silencing of CCNA2 by sequestering EZH2 and preventing H3K27me3 accumulation at the CCNA2 PRE-like element. Thereby, G 0 cells are held in suspended animation, poised to return to active proliferation, with a subsequent option to differentiate if conditions are conducive. PRDM2 knockdown permits EZH2 to accumulate at the PRE, leading to increased H3K27me3, silencing CCNA2 and diverting quiescence toward differentiation.

    Techniques Used: Genome Wide, Preserving, Methylation

    A PRE-like element in the CCNA2 bivalent domain shows cell state-specific EZH1 and EZH2 binding that is regulated by PRDM2. ( A ) Schematic of CCNA2 locus: exons-red arrows; introns-thin red lines. YY1 binding sites (GCCATHWY), PHO binding sites (CNGCCATNDNND) and GAF binding sites (GGGAAGG, GAGGGG, GAGAG) are clustered in two distinct regions in introns 1 and 2 (Region 1 and 2) respectively). ( B ) Cloned Regions 1 and 2 repress luciferase reporter activity in transient transfection assays, compared to empty vector control. Positive control for repressor activity is a known repressive element from EVX2–HoxD13 region; negative control is GAPDH gene (500 bp). Values represent normalized reporter activity (mean ± S.D., n = 3; * P
    Figure Legend Snippet: A PRE-like element in the CCNA2 bivalent domain shows cell state-specific EZH1 and EZH2 binding that is regulated by PRDM2. ( A ) Schematic of CCNA2 locus: exons-red arrows; introns-thin red lines. YY1 binding sites (GCCATHWY), PHO binding sites (CNGCCATNDNND) and GAF binding sites (GGGAAGG, GAGGGG, GAGAG) are clustered in two distinct regions in introns 1 and 2 (Region 1 and 2) respectively). ( B ) Cloned Regions 1 and 2 repress luciferase reporter activity in transient transfection assays, compared to empty vector control. Positive control for repressor activity is a known repressive element from EVX2–HoxD13 region; negative control is GAPDH gene (500 bp). Values represent normalized reporter activity (mean ± S.D., n = 3; * P

    Techniques Used: Binding Assay, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Positive Control, Negative Control

    16) Product Images from "Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE"

    Article Title: Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.007

    HOXA11-AS Can Recruit EZH2 and LSD1 to Silence RND3 Expression (A) Cell fractionation assays indicated that HOXA11-AS is mostly located in the nucleus. GAPDH and U1 acted as markers of the cytoplasm and nucleus, respectively. (B) Bioinformatics were used to predict the possibility of interaction of HOXA11-AS . Predictions with probabilities > 0.5 were considered positive. RPISeq predictions are based on random forest (RF) or support vector machine (SVM). (C) RIP experiments were performed, and the coprecipitated RNA was detected by qRT-PCR. (D) In vitro -transcribed pulldown assays showed that HOXA11-AS could retrieve EZH2 and LSD1 in HTR-8/SVneo cells but not G9a. G9a was a negative control. (E) Western blot assays detected the expression of RND3 after silenced EZH2 after si-RNA transfection in HTR-8/SVneo cells. (F) The enrichment of EZH2 /H3K27me3 and LSD1 /H3K4me2 in the promoter regions of RND3 was identified via ChIP assays, and this enrichment was decreased after HOXA11-AS knockdown in the HTR-8/SVneo cell line. Antibody directed against immunoglobulin G (IgG) was used as a negative control. Values represent the mean ± SEM from three independent experiments. **p
    Figure Legend Snippet: HOXA11-AS Can Recruit EZH2 and LSD1 to Silence RND3 Expression (A) Cell fractionation assays indicated that HOXA11-AS is mostly located in the nucleus. GAPDH and U1 acted as markers of the cytoplasm and nucleus, respectively. (B) Bioinformatics were used to predict the possibility of interaction of HOXA11-AS . Predictions with probabilities > 0.5 were considered positive. RPISeq predictions are based on random forest (RF) or support vector machine (SVM). (C) RIP experiments were performed, and the coprecipitated RNA was detected by qRT-PCR. (D) In vitro -transcribed pulldown assays showed that HOXA11-AS could retrieve EZH2 and LSD1 in HTR-8/SVneo cells but not G9a. G9a was a negative control. (E) Western blot assays detected the expression of RND3 after silenced EZH2 after si-RNA transfection in HTR-8/SVneo cells. (F) The enrichment of EZH2 /H3K27me3 and LSD1 /H3K4me2 in the promoter regions of RND3 was identified via ChIP assays, and this enrichment was decreased after HOXA11-AS knockdown in the HTR-8/SVneo cell line. Antibody directed against immunoglobulin G (IgG) was used as a negative control. Values represent the mean ± SEM from three independent experiments. **p

    Techniques Used: Expressing, Cell Fractionation, Plasmid Preparation, Quantitative RT-PCR, In Vitro, Negative Control, Western Blot, Transfection, Chromatin Immunoprecipitation

    17) Product Images from "Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells"

    Article Title: Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.01.013

    linc00665 interacts with EZH2 to regulate CDKN1C in NSCLC cells (A) Quantitative real-time PCR was used to detect the relative expression of linc00665 in the cytoplasm and nucleus of H1299 and H1975 cell lines. GAPDH was used as a cytoplasmic marker; U6 was used as a nuclear marker. (B) Using RNA immunoprecipitation analysis and qRT-PCR to detect the expression level of linc00665 RNA, we compared the immunoprecipitation of LSD1, EZH2, and SUZ12 with that of IgG. (C) qRT-PCR was used to detect the expression level of tumor suppressor in NSCLC cells transfected with si-linc00665. (D) CDKN1C protein levels in linc00665 knockdown H1299 and H1975 cells transfected with si-linc00665 #2 or si-linc00665 #4. (E and F) mRNA and protein levels of EZH2/LSD1 and CDKN1C in H1975 and H1299 cells transfected with si-EZH2 and si-LSD1 were detected by qRT-PCR and western blot analysis. (G) The binding ratio of H3K27me3 to EZH2 in the CDKN1C initiating region in H1299 and H1975 cells transfected with si-linc00665 was determined by a ChIP assay. (H) The expression of EZH2 was detected after linc00665 was knocked down. ∗p
    Figure Legend Snippet: linc00665 interacts with EZH2 to regulate CDKN1C in NSCLC cells (A) Quantitative real-time PCR was used to detect the relative expression of linc00665 in the cytoplasm and nucleus of H1299 and H1975 cell lines. GAPDH was used as a cytoplasmic marker; U6 was used as a nuclear marker. (B) Using RNA immunoprecipitation analysis and qRT-PCR to detect the expression level of linc00665 RNA, we compared the immunoprecipitation of LSD1, EZH2, and SUZ12 with that of IgG. (C) qRT-PCR was used to detect the expression level of tumor suppressor in NSCLC cells transfected with si-linc00665. (D) CDKN1C protein levels in linc00665 knockdown H1299 and H1975 cells transfected with si-linc00665 #2 or si-linc00665 #4. (E and F) mRNA and protein levels of EZH2/LSD1 and CDKN1C in H1975 and H1299 cells transfected with si-EZH2 and si-LSD1 were detected by qRT-PCR and western blot analysis. (G) The binding ratio of H3K27me3 to EZH2 in the CDKN1C initiating region in H1299 and H1975 cells transfected with si-linc00665 was determined by a ChIP assay. (H) The expression of EZH2 was detected after linc00665 was knocked down. ∗p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Marker, Immunoprecipitation, Quantitative RT-PCR, Transfection, Western Blot, Binding Assay, Chromatin Immunoprecipitation

    18) Product Images from "Overexpressed long noncoding RNA TUG1 affects the cell cycle, proliferation, and apoptosis of pancreatic cancer partly through suppressing RND3 and MT2A"

    Article Title: Overexpressed long noncoding RNA TUG1 affects the cell cycle, proliferation, and apoptosis of pancreatic cancer partly through suppressing RND3 and MT2A

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S188396

    TUG1 interacted with EZH2 to repress the expression of RND3 and MT2A. Notes: ( A ) Relative levels of TUG1 in the cell cytoplasm or nucleus of AsPC-1 and BxPC-3 cells were determined using qPCR. GAPDH was used as cytoplasmic control and U1 was used as nuclear control. The distribution of TUG1 RNA in the cytoplasm or nucleus was represented as the percentage rate of total RNA. ( B ) The RNA levels in immunoprecipitates with EZH2 and SUZ12 were determined using qPCR. The expression levels of TUG1 RNA are represented as fold enrichment relative to the IgG immunoprecipitate. ( C ) The relative expression levels of EZH2 in AsPC-1 and BxPC-3 cells transfected with si-NC or si-EZH2 (si-EZH2 1#, si-EZH2 2#, and si-EZH2 3#) were measured using qPCR. ( D ) The mRNA levels of RND3, MT2A, KLF2, PLK3, P15, P21, LATS1, LATS2, RRAD, and ASPP2 were determined using qPCR after knockdown of EZH2 in AsPC-1 and BxPC-3 cells. ( E ) ChIP shows EZH2/H3K27me3 occupancy on the RND3 or MT2A promoter regions in AsPc-1 and BxPc-3 cells, and knockdown of AGAP2-AS1 decreases their occupancy. The mean values and SD were calculated from triplicates of a representative experiment. * P
    Figure Legend Snippet: TUG1 interacted with EZH2 to repress the expression of RND3 and MT2A. Notes: ( A ) Relative levels of TUG1 in the cell cytoplasm or nucleus of AsPC-1 and BxPC-3 cells were determined using qPCR. GAPDH was used as cytoplasmic control and U1 was used as nuclear control. The distribution of TUG1 RNA in the cytoplasm or nucleus was represented as the percentage rate of total RNA. ( B ) The RNA levels in immunoprecipitates with EZH2 and SUZ12 were determined using qPCR. The expression levels of TUG1 RNA are represented as fold enrichment relative to the IgG immunoprecipitate. ( C ) The relative expression levels of EZH2 in AsPC-1 and BxPC-3 cells transfected with si-NC or si-EZH2 (si-EZH2 1#, si-EZH2 2#, and si-EZH2 3#) were measured using qPCR. ( D ) The mRNA levels of RND3, MT2A, KLF2, PLK3, P15, P21, LATS1, LATS2, RRAD, and ASPP2 were determined using qPCR after knockdown of EZH2 in AsPC-1 and BxPC-3 cells. ( E ) ChIP shows EZH2/H3K27me3 occupancy on the RND3 or MT2A promoter regions in AsPc-1 and BxPc-3 cells, and knockdown of AGAP2-AS1 decreases their occupancy. The mean values and SD were calculated from triplicates of a representative experiment. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation

    19) Product Images from "Abnormally glycosylated MUC1 establishes a positive feedback circuit of inflammatory cytokines, mediated by NF-κB p65 and EzH2, in colitis-associated cancer"

    Article Title: Abnormally glycosylated MUC1 establishes a positive feedback circuit of inflammatory cytokines, mediated by NF-κB p65 and EzH2, in colitis-associated cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22168

    EzH2 regulates inflammatory cytokines expression in MUC1+ IECs from AOM/DSS-treated mice (A) Confocal immunofluorescence microscopy of frozen colon tissue samples, fixed and stained with anti-NF-κB p65 (red) or EzH2 (green) antibodies. Bar: 100 μm. (B) Co-Immunoprecipitation Assay. NF-kB, p65 immunoprecipitated nuclear proteins from indicated cells were immunoblotted with anti-EzH2 antibody. (C-D) ChIP assay: soluble chromatin was immunoprecipitated with indicated antibodies and analyzed for κB consensus sites of IL-6 and TNF-α promoters. Quantification of binding was represented as fold-enrichment relative to IgG. (E) Western blotting of whole cell lysates of IECs transfected with an shRNA targeting EzH2 (shEzH2) or a target control (shNegCont), as described in Material and Methods, with anti-EzH2 antibody. Actin was used as loading control. (F) Indicated cytokine production by IECs transfected with shEzH2 RNA or target control (shNegCont). N=6 per group. Statistical analysis was carried out with one-way ANOVA, * indicates P
    Figure Legend Snippet: EzH2 regulates inflammatory cytokines expression in MUC1+ IECs from AOM/DSS-treated mice (A) Confocal immunofluorescence microscopy of frozen colon tissue samples, fixed and stained with anti-NF-κB p65 (red) or EzH2 (green) antibodies. Bar: 100 μm. (B) Co-Immunoprecipitation Assay. NF-kB, p65 immunoprecipitated nuclear proteins from indicated cells were immunoblotted with anti-EzH2 antibody. (C-D) ChIP assay: soluble chromatin was immunoprecipitated with indicated antibodies and analyzed for κB consensus sites of IL-6 and TNF-α promoters. Quantification of binding was represented as fold-enrichment relative to IgG. (E) Western blotting of whole cell lysates of IECs transfected with an shRNA targeting EzH2 (shEzH2) or a target control (shNegCont), as described in Material and Methods, with anti-EzH2 antibody. Actin was used as loading control. (F) Indicated cytokine production by IECs transfected with shEzH2 RNA or target control (shNegCont). N=6 per group. Statistical analysis was carried out with one-way ANOVA, * indicates P

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Microscopy, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay, Western Blot, Transfection, shRNA

    AOM/DSS-treatment up-regulated EzH2 expression via NF-κB pathway activation in IECs of MUC1.Tg (A) Eighty-four cancer related genes were analyzed using RT 2 Profiler™ PCR Array. Scatter plot of the hybridization intensity of each gene in the two groups: WT (x-axis-group 1) and MUC1.Tg (y-axis- group 2). The middle line indicates a fold-change (2-ΔCt) of 1. The top and the bottom lines indicate the fold-change in gene expression threshold. The two points under the bottom line represent downregulated genes. Significant genes with fold change
    Figure Legend Snippet: AOM/DSS-treatment up-regulated EzH2 expression via NF-κB pathway activation in IECs of MUC1.Tg (A) Eighty-four cancer related genes were analyzed using RT 2 Profiler™ PCR Array. Scatter plot of the hybridization intensity of each gene in the two groups: WT (x-axis-group 1) and MUC1.Tg (y-axis- group 2). The middle line indicates a fold-change (2-ΔCt) of 1. The top and the bottom lines indicate the fold-change in gene expression threshold. The two points under the bottom line represent downregulated genes. Significant genes with fold change

    Techniques Used: Expressing, Activation Assay, Polymerase Chain Reaction, Hybridization

    Co-expression of hypoglycosylated MUC1 with p-p65 and EzH2 in colon adenocarcinoma (A) Immunohistochemistry staining of the indicated proteins in colon adenocarcinoma. Original magnification is 200X. (B) Quantification of the percent of colon adenocarcinoma expressing total MUC1 (HMPV), hypoglycosylated MUC1 (VU-4H5), p-p65 (276) and EzH2 in colon adenocarcinoma (n=70).
    Figure Legend Snippet: Co-expression of hypoglycosylated MUC1 with p-p65 and EzH2 in colon adenocarcinoma (A) Immunohistochemistry staining of the indicated proteins in colon adenocarcinoma. Original magnification is 200X. (B) Quantification of the percent of colon adenocarcinoma expressing total MUC1 (HMPV), hypoglycosylated MUC1 (VU-4H5), p-p65 (276) and EzH2 in colon adenocarcinoma (n=70).

    Techniques Used: Expressing, Immunohistochemistry, Staining

    20) Product Images from "MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines *"

    Article Title: MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.750950

    Knockdown of MEG3 affected the TGF-β-induced regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with retroviruses expressing control shRNA or MEG3 shRNA#1 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (*, p
    Figure Legend Snippet: Knockdown of MEG3 affected the TGF-β-induced regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with retroviruses expressing control shRNA or MEG3 shRNA#1 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (*, p

    Techniques Used: Methylation, Infection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Knockdown of JARID2 inhibited MEG3 -induced changes in the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p
    Figure Legend Snippet: Knockdown of JARID2 inhibited MEG3 -induced changes in the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 and/or the retrovirus expressing JARID2 shRNA without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p

    Techniques Used: Methylation, Infection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Overexpression of MEG3 affected the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p
    Figure Legend Snippet: Overexpression of MEG3 affected the regulation of histone H3 methylation and EZH2 recruitment on the regulatory regions of CDH1 gene and miR-200 gene clusters in A549 or LC-2/ad cells. A549 or LC-2/ad cells were infected with the control retrovirus or the retrovirus expressing MEG3 without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and H3K4me3 on the regulatory regions of CDH1 ( A and E ), miR-200b/200a/429 ( B and F ), miR-200c/141 ( C and G ), and GAPDH genes ( D and H ) in A549 ( A–D ) or LC-2/ad ( E–H ) cells are shown. The occupancies of methylated histones or EZH2 protein on the regions were analyzed by quantitative PCR (*, p

    Techniques Used: Over Expression, Methylation, Infection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    MEG3 interacted with JARID2 and stimulated the interaction and recruitment of JARID2 and EZH2 on the regulatory regions of CDH1 , miR-200a, and miR-200c genes for histone H3K27 methylation in A549 cells. A, interaction of MEG3 and JARID2 detected by RIP. A549 cells were infected with the various combinations (as indicated) of the retroviruses expressing MEG3 and FLAG-tagged JARID2. The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG) or anti-FLAG antibody, and the co-precipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 in the precipitates. n.d. means not detected. B–E, A549 cells were infected with the various combinations (as indicated) of the retroviruses without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and FLAG-tagged JARID2 on the regulatory regions of CDH1 ( B ), miR-200b/200a/429 ( C ), miR-200c/141 ( D ), and GAPDH genes ( E ) are shown (*, p
    Figure Legend Snippet: MEG3 interacted with JARID2 and stimulated the interaction and recruitment of JARID2 and EZH2 on the regulatory regions of CDH1 , miR-200a, and miR-200c genes for histone H3K27 methylation in A549 cells. A, interaction of MEG3 and JARID2 detected by RIP. A549 cells were infected with the various combinations (as indicated) of the retroviruses expressing MEG3 and FLAG-tagged JARID2. The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG) or anti-FLAG antibody, and the co-precipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 in the precipitates. n.d. means not detected. B–E, A549 cells were infected with the various combinations (as indicated) of the retroviruses without or with TGF-β treatment. ChIP analyses of H3K27me3, EZH2, and FLAG-tagged JARID2 on the regulatory regions of CDH1 ( B ), miR-200b/200a/429 ( C ), miR-200c/141 ( D ), and GAPDH genes ( E ) are shown (*, p

    Techniques Used: Methylation, Infection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    21) Product Images from "A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma"

    Article Title: A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03447-8

    Sensitivity to EZH2 inhibition was associated with DNA damage, G2 arrest and apoptosis. a – b Flow cytometry analysis of apoptotic markers in ( a ) INA-6 and ( b ) U1996 cells upon UNC1999 treatment. c Representative western blot against γH2AX in INA-6 and U1996 cells and signal quantification of the western blot. Optical density was normalized against H4. The corresponding uncropped western blots can be found in Supplementary Fig. 9d . d – e Flow cytometry analysis of the cell cycle in ( d ) INA-6 and ( e ) U1996 cells upon UNC1999 treatment. Statistical analysis was performed with one-way ANOVA and multiple t-test. All experiments were performed in three biological replicates. Values: mean with SEM. * p
    Figure Legend Snippet: Sensitivity to EZH2 inhibition was associated with DNA damage, G2 arrest and apoptosis. a – b Flow cytometry analysis of apoptotic markers in ( a ) INA-6 and ( b ) U1996 cells upon UNC1999 treatment. c Representative western blot against γH2AX in INA-6 and U1996 cells and signal quantification of the western blot. Optical density was normalized against H4. The corresponding uncropped western blots can be found in Supplementary Fig. 9d . d – e Flow cytometry analysis of the cell cycle in ( d ) INA-6 and ( e ) U1996 cells upon UNC1999 treatment. Statistical analysis was performed with one-way ANOVA and multiple t-test. All experiments were performed in three biological replicates. Values: mean with SEM. * p

    Techniques Used: Inhibition, Flow Cytometry, Western Blot

    Schematic representation of the downstream effects of EZH2i on the metabolic profile of sensitive MM cell lines. A UNC1999 inhibits the activity of EZH2. B This prevents EZH2 from methylating H3, thus resulting in loss of H3K27me3 and open chromatin. As a consequence, the tumour-suppressor miRNAs miR-494-39, miR-192-5p, miR-130a-3p and miR-134-5p, which are silenced by H3K27me3 under basal conditions, are upregulated. C The tumour-suppressor miRNAs downregulate their target genes ( MAT2A , MAT2B , CBS and CTH ), which encode for enzymes involved in methionine cycling. This dysregulates methionine cycling and results in variation of metabolite abundance, such as accumulation of homocysteine. Metabolites in each pathway are indicated by small circles, which are colour coded with the same colour of the pathway they participate in. Metabolites that vary in abundance post UNC1999 treatment are marked by either a red (decreased abundance) or a green (accumulation) border.
    Figure Legend Snippet: Schematic representation of the downstream effects of EZH2i on the metabolic profile of sensitive MM cell lines. A UNC1999 inhibits the activity of EZH2. B This prevents EZH2 from methylating H3, thus resulting in loss of H3K27me3 and open chromatin. As a consequence, the tumour-suppressor miRNAs miR-494-39, miR-192-5p, miR-130a-3p and miR-134-5p, which are silenced by H3K27me3 under basal conditions, are upregulated. C The tumour-suppressor miRNAs downregulate their target genes ( MAT2A , MAT2B , CBS and CTH ), which encode for enzymes involved in methionine cycling. This dysregulates methionine cycling and results in variation of metabolite abundance, such as accumulation of homocysteine. Metabolites in each pathway are indicated by small circles, which are colour coded with the same colour of the pathway they participate in. Metabolites that vary in abundance post UNC1999 treatment are marked by either a red (decreased abundance) or a green (accumulation) border.

    Techniques Used: Activity Assay

    EZH2 inhibition significantly reduced tumour burden in mice bearing 5T33MM multiple myeloma. a Western blot against H3K27me3 and total histone H3 (H3) of bone marrow plasma cells derived from 5T33MM mice treated with 150 or 300 mg/kg of UNC1999, or with vehicle. Total histone H4 (H4) was used as a loading control. Corresponding uncropped western blots can be found in Supplementary Fig. 9a–b . b Signal quantification of the western blot against H3K27me3 shown in ( a ). c Signal quantification of the western blot against H3 shown in ( a ). The optical density in ( b – c ) was normalized against H4. n (Vehicle) = 4, n (150 mg/kg) = 5 and n (300 mg/kg) = 4. Statistical analysis in ( b – c ) was performed with one-way ANOVA. Values: mean with SEM. d – f 5T33MM mice were treated with 200 mg/kg of UNC1999 or with vehicle. Tumour load was determined by d bone marrow plasmacytosis, e M-spike levels and f spleen weight. n (Vehicle) = 8, n (UNC1999) = 6. Statistical analysis in ( d – f ) was performed with one-sided t -test. Values: mean with SEM. * p
    Figure Legend Snippet: EZH2 inhibition significantly reduced tumour burden in mice bearing 5T33MM multiple myeloma. a Western blot against H3K27me3 and total histone H3 (H3) of bone marrow plasma cells derived from 5T33MM mice treated with 150 or 300 mg/kg of UNC1999, or with vehicle. Total histone H4 (H4) was used as a loading control. Corresponding uncropped western blots can be found in Supplementary Fig. 9a–b . b Signal quantification of the western blot against H3K27me3 shown in ( a ). c Signal quantification of the western blot against H3 shown in ( a ). The optical density in ( b – c ) was normalized against H4. n (Vehicle) = 4, n (150 mg/kg) = 5 and n (300 mg/kg) = 4. Statistical analysis in ( b – c ) was performed with one-way ANOVA. Values: mean with SEM. d – f 5T33MM mice were treated with 200 mg/kg of UNC1999 or with vehicle. Tumour load was determined by d bone marrow plasmacytosis, e M-spike levels and f spleen weight. n (Vehicle) = 8, n (UNC1999) = 6. Statistical analysis in ( d – f ) was performed with one-sided t -test. Values: mean with SEM. * p

    Techniques Used: Inhibition, Mouse Assay, Western Blot, Derivative Assay

    22) Product Images from "β-Arrestin1 promotes the progression of chronic myeloid leukaemia by regulating BCR/ABL H4 acetylation"

    Article Title: β-Arrestin1 promotes the progression of chronic myeloid leukaemia by regulating BCR/ABL H4 acetylation

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.335

    β -Arrestin1 interacts with EZH2 in the nucleus of K562 cells. ( A ) The expression of EZH2 in stable K562-si β 1 and K562-Ctrl cells was measured by WB with β -actin as the loading control. ( B ) The expression levels of H3K27me3 were analysed by WB, and H3 as the control. ( C ) K562 nuclear extracts were immunoprecipitated with EZH2 or β -arrestin1 antibody and the immunocomplexes were analysed in WB using antibodies against EZH2 or β -arrestin1. IgG as the negative control. ( D ) Confocal visualisation of EZH2 (green) and β -arrestin1 (red) in different stable K562 cells. And single confocal sections were shown. Scale bar, 5 μ m. The full colour version of this figure is available at British Journal of Cancer online.
    Figure Legend Snippet: β -Arrestin1 interacts with EZH2 in the nucleus of K562 cells. ( A ) The expression of EZH2 in stable K562-si β 1 and K562-Ctrl cells was measured by WB with β -actin as the loading control. ( B ) The expression levels of H3K27me3 were analysed by WB, and H3 as the control. ( C ) K562 nuclear extracts were immunoprecipitated with EZH2 or β -arrestin1 antibody and the immunocomplexes were analysed in WB using antibodies against EZH2 or β -arrestin1. IgG as the negative control. ( D ) Confocal visualisation of EZH2 (green) and β -arrestin1 (red) in different stable K562 cells. And single confocal sections were shown. Scale bar, 5 μ m. The full colour version of this figure is available at British Journal of Cancer online.

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Negative Control

    DZNep inhibits β -arrestin1-EZH2 binding, alters BCR/ABL gene expression and acetylation and further suppresses K562 cells progression. ( A ) K562 cells were pretreated with or without 2 μ M DZNep for 24 h. And then Co-IP was performed to detect the expression of EZH2 and its interaction with β -arrestin1. ( B ) Different K562 cells were cultured in a two-layer soft agar for 14 days, and the colonies (⩾40 cells for each) were counted. Experiments were performed triplicate at each point. ( C ) Stable K562-Ctrl cells were injected into the tail vein of lethally irradiated NOD/SCID mice. Ten days later, DZNep was intraperitoneally injected at a dose of 2 mg kg −1 daily and the survival of mice was observed. The control mice received PBS. n =10 each group. ( D ) ChIP analysis was used to determine the effects of DZNep in the acetylation of BCR and ABL. ( E ) The expression of BCR/ABL quantified by real-time RT–PCR (lower panel) and WB (upper panel). ( F ) The scheme of this study. * P
    Figure Legend Snippet: DZNep inhibits β -arrestin1-EZH2 binding, alters BCR/ABL gene expression and acetylation and further suppresses K562 cells progression. ( A ) K562 cells were pretreated with or without 2 μ M DZNep for 24 h. And then Co-IP was performed to detect the expression of EZH2 and its interaction with β -arrestin1. ( B ) Different K562 cells were cultured in a two-layer soft agar for 14 days, and the colonies (⩾40 cells for each) were counted. Experiments were performed triplicate at each point. ( C ) Stable K562-Ctrl cells were injected into the tail vein of lethally irradiated NOD/SCID mice. Ten days later, DZNep was intraperitoneally injected at a dose of 2 mg kg −1 daily and the survival of mice was observed. The control mice received PBS. n =10 each group. ( D ) ChIP analysis was used to determine the effects of DZNep in the acetylation of BCR and ABL. ( E ) The expression of BCR/ABL quantified by real-time RT–PCR (lower panel) and WB (upper panel). ( F ) The scheme of this study. * P

    Techniques Used: Binding Assay, Expressing, Co-Immunoprecipitation Assay, Cell Culture, Injection, Irradiation, Mouse Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Western Blot

    23) Product Images from "Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1 replication by displacing the polycomb repressive complex 2 from binding to the LTR promoter"

    Article Title: Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1 replication by displacing the polycomb repressive complex 2 from binding to the LTR promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz117

    MALAT1 knockout promotes EZH2 binding to HIV-1 LTR and increases the modification of H3K27me3. ( A ) A schematic illustration of the interaction between MALAT1 and PRC2 subunits. ( B ) Associations of EZH2, SUZ12 and EED with MALAT1 in Jurkat T cells as determined with RIP assay. ( C ) A schematic illustration of nucleosomes on HIV-1 5′-LTR region. ( D – F ) MALAT1 knockout promotes EZH2 binding to HIV-1 LTR and increases the modification of H3K27me3. MALAT1-stably-knocking-out HEK293T (D) or Jurkat T cells (E), were infected with HIV-luc/VSV-G for 2 days, the associations of EZH2 (D, E, left panels), H3K27me3 (D, E, right panels) and H3K9me3 (F) with HIV-1 5′-LTR were determined by a cross-linked ChIP assay. Data were presented as mean ± SD. Results were representative of at least three independent experiments. ** P
    Figure Legend Snippet: MALAT1 knockout promotes EZH2 binding to HIV-1 LTR and increases the modification of H3K27me3. ( A ) A schematic illustration of the interaction between MALAT1 and PRC2 subunits. ( B ) Associations of EZH2, SUZ12 and EED with MALAT1 in Jurkat T cells as determined with RIP assay. ( C ) A schematic illustration of nucleosomes on HIV-1 5′-LTR region. ( D – F ) MALAT1 knockout promotes EZH2 binding to HIV-1 LTR and increases the modification of H3K27me3. MALAT1-stably-knocking-out HEK293T (D) or Jurkat T cells (E), were infected with HIV-luc/VSV-G for 2 days, the associations of EZH2 (D, E, left panels), H3K27me3 (D, E, right panels) and H3K9me3 (F) with HIV-1 5′-LTR were determined by a cross-linked ChIP assay. Data were presented as mean ± SD. Results were representative of at least three independent experiments. ** P

    Techniques Used: Knock-Out, Binding Assay, Modification, Stable Transfection, Infection, Chromatin Immunoprecipitation

    MALAT1 antagonizes EZH2-mediated silencing of viral gene transcription to promote HIV-1 replication. ( A–C ) MALAT1 restores HIV-1 infection by antagonizing EZH2-mediated inhibition. MALAT1-stably-knocking-out HEK293T cells were infected with lentivirus containing EZH2 specific shRNA or off-target controls for 48 h to further knockdown EZH2 expression, cells were then infected with HIV-luc/VSV-G for an additional 24 h. EZH2 knockdown was detected by RT-PCR (A) and western blot (B). Viral infection was detected by quantifying cell-associated HIV-1 gag mRNA (C). ( D and E ) EZH2 expression in primary CD4 + T cells. Resting CD4 + T cells (1 × 10 6 ) were stimulated with or without PHA-P (5 μg/ml) for 3 days, and the endogenous expression of EZH2 was detected by either RT-PCR (D) or western blot (E). ( F – H ) The double knockdown of MALAT1 and EZH2 rescues HIV-1 infection in primary CD4 + T cells. PHA-P-activated primary CD4 + T cells (1 × 10 6 ) were infected with lentiviruses containing MALAT1 or/and EZH2-specific shRNA or off-target controls for 48 h, then cells were infected with replication-competent HIV-1 NL4-3 for an additional 96 h. MALAT1 knockdown was determined by RT-PCR (F); the expression of endogenous EZH2 was detected by RT-PCR and western blot (G); viral replication was quantified by detecting p24 gag levels in the supernatants by ELISA (H). Data were presented as mean ± SD. Results were representative of three independent experiments. * P
    Figure Legend Snippet: MALAT1 antagonizes EZH2-mediated silencing of viral gene transcription to promote HIV-1 replication. ( A–C ) MALAT1 restores HIV-1 infection by antagonizing EZH2-mediated inhibition. MALAT1-stably-knocking-out HEK293T cells were infected with lentivirus containing EZH2 specific shRNA or off-target controls for 48 h to further knockdown EZH2 expression, cells were then infected with HIV-luc/VSV-G for an additional 24 h. EZH2 knockdown was detected by RT-PCR (A) and western blot (B). Viral infection was detected by quantifying cell-associated HIV-1 gag mRNA (C). ( D and E ) EZH2 expression in primary CD4 + T cells. Resting CD4 + T cells (1 × 10 6 ) were stimulated with or without PHA-P (5 μg/ml) for 3 days, and the endogenous expression of EZH2 was detected by either RT-PCR (D) or western blot (E). ( F – H ) The double knockdown of MALAT1 and EZH2 rescues HIV-1 infection in primary CD4 + T cells. PHA-P-activated primary CD4 + T cells (1 × 10 6 ) were infected with lentiviruses containing MALAT1 or/and EZH2-specific shRNA or off-target controls for 48 h, then cells were infected with replication-competent HIV-1 NL4-3 for an additional 96 h. MALAT1 knockdown was determined by RT-PCR (F); the expression of endogenous EZH2 was detected by RT-PCR and western blot (G); viral replication was quantified by detecting p24 gag levels in the supernatants by ELISA (H). Data were presented as mean ± SD. Results were representative of three independent experiments. * P

    Techniques Used: Infection, Inhibition, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    A schematic illustration of MALAT1 activates HIV-1 replication by displacing PRC2 from binding to the LTR and preventing it from mediating epigenetic silencing. The association of MALAT1 with PRC2 detaches the core component EZH2 from binding to HIV-1 LTR promoter, thus preventing epigenetic silencing caused by EZH2-mediated H3K27me3 methylation of HIV-1 LTR regions.
    Figure Legend Snippet: A schematic illustration of MALAT1 activates HIV-1 replication by displacing PRC2 from binding to the LTR and preventing it from mediating epigenetic silencing. The association of MALAT1 with PRC2 detaches the core component EZH2 from binding to HIV-1 LTR promoter, thus preventing epigenetic silencing caused by EZH2-mediated H3K27me3 methylation of HIV-1 LTR regions.

    Techniques Used: Binding Assay, Methylation

    LRAs induce MALAT1 expression in HIV-1 latently infected cells. HIV-1 latently infected CD4 + CEM cells (ACH2) ( A ) and Jurkat T-cell (C11 clone) ( C–E ) were stimulated with PMA/Ionomycin at indicated concentrations for 24 h, and viral reactivation was detected by quantifying the cell-associated gag mRNA (A, middle panel; D), or titrating the produced viral particles in TZMB1 indicator cells (A, right panel), or detecting GFP expression (E). The associations of EZH2 with HIV-1 5′-LTR were determined by a cross-linked ChIP assay (B and F ). ( G ) MALAT1 expression by stimulation with SAHA. ACH2 or C11 were treated with SAHA for 24 h, viral reactivation was detected by quantifying cell-associated HIV-1 gag mRNA. MALAT1 expression was quantified by RT-PCR and normalized with GAPDH (A, C and G). Results were representative of three independent experiments. * P
    Figure Legend Snippet: LRAs induce MALAT1 expression in HIV-1 latently infected cells. HIV-1 latently infected CD4 + CEM cells (ACH2) ( A ) and Jurkat T-cell (C11 clone) ( C–E ) were stimulated with PMA/Ionomycin at indicated concentrations for 24 h, and viral reactivation was detected by quantifying the cell-associated gag mRNA (A, middle panel; D), or titrating the produced viral particles in TZMB1 indicator cells (A, right panel), or detecting GFP expression (E). The associations of EZH2 with HIV-1 5′-LTR were determined by a cross-linked ChIP assay (B and F ). ( G ) MALAT1 expression by stimulation with SAHA. ACH2 or C11 were treated with SAHA for 24 h, viral reactivation was detected by quantifying cell-associated HIV-1 gag mRNA. MALAT1 expression was quantified by RT-PCR and normalized with GAPDH (A, C and G). Results were representative of three independent experiments. * P

    Techniques Used: Expressing, Infection, Produced, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Disruption Of MYC-MiRNA-EZH2 Loop To Suppress Aggressive B-Cell Lymphoma Survival And Clonogenicity"

    Article Title: Disruption Of MYC-MiRNA-EZH2 Loop To Suppress Aggressive B-Cell Lymphoma Survival And Clonogenicity

    Journal: Leukemia

    doi: 10.1038/leu.2013.94

    MiR-26a is co-regulated by MYC and EZH2
    Figure Legend Snippet: MiR-26a is co-regulated by MYC and EZH2

    Techniques Used:

    MYC recruits EZH2 to miR-26a promoters to repress miR-26a expression in lymphoma cell lines and primary lymphoma cells
    Figure Legend Snippet: MYC recruits EZH2 to miR-26a promoters to repress miR-26a expression in lymphoma cell lines and primary lymphoma cells

    Techniques Used: Expressing

    MiR-26a is required for the effect of MYC and EZH2 on lymphoma cell growth
    Figure Legend Snippet: MiR-26a is required for the effect of MYC and EZH2 on lymphoma cell growth

    Techniques Used:

    Silencing MYC with JQ1 disrupts the MYC-miRNA-EZH2 regulatory circuitry and suppresses tumor cell growth and clonogenecity
    Figure Legend Snippet: Silencing MYC with JQ1 disrupts the MYC-miRNA-EZH2 regulatory circuitry and suppresses tumor cell growth and clonogenecity

    Techniques Used:

    25) Product Images from "Hyperoxia-induced methylation decreases RUNX3 in a newborn rat model of bronchopulmonary dysplasia"

    Article Title: Hyperoxia-induced methylation decreases RUNX3 in a newborn rat model of bronchopulmonary dysplasia

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0239-x

    EZH2 protein and mRNA levels in lung tissues and AT2 cells following hyperoxia. In the model group, EZH2 protein, as determined by Western blot, ( a and b ) and mRNA, as determined by real time-PCR ( c ), were highly expressed in lung tissues after 10 day of hyperoxia, while EZH2 protein ( d and e ) and mRNA ( f ) were highly expressed in AT2 cells after 7 days of hyperoxia. C: Control group, E: Model group, * P
    Figure Legend Snippet: EZH2 protein and mRNA levels in lung tissues and AT2 cells following hyperoxia. In the model group, EZH2 protein, as determined by Western blot, ( a and b ) and mRNA, as determined by real time-PCR ( c ), were highly expressed in lung tissues after 10 day of hyperoxia, while EZH2 protein ( d and e ) and mRNA ( f ) were highly expressed in AT2 cells after 7 days of hyperoxia. C: Control group, E: Model group, * P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction

    Binding between H3K27me3/EZH2 and the RUNX3 promoter in AT2 cells. As determined by ChIP, binding levels between H3K27me3 ( a ) or EZH2 ( b ) and the RUNX3 promoter region were higher in AT2 cells after 14 days of hyperoxia compared to the control group. Gene locus 1: RUNX3 promoter region −5407 ~ −5142 bp; 2: RUNX3 promoter region −5407 ~ −5215 bp; 3: RUNX3 promoter region −5407 ~ −5142 bp. * P
    Figure Legend Snippet: Binding between H3K27me3/EZH2 and the RUNX3 promoter in AT2 cells. As determined by ChIP, binding levels between H3K27me3 ( a ) or EZH2 ( b ) and the RUNX3 promoter region were higher in AT2 cells after 14 days of hyperoxia compared to the control group. Gene locus 1: RUNX3 promoter region −5407 ~ −5142 bp; 2: RUNX3 promoter region −5407 ~ −5215 bp; 3: RUNX3 promoter region −5407 ~ −5142 bp. * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    Analysis of the correlation between RUNX3 and DNMTs or EZH2 protein expression in AT2 cells. RUNX3 protein expression correlated positively with DNMT1 protein expression ( a ), but negatively with DNMT3b ( b ) and EZH2 ( c ) protein expression in AT2 cells. * P
    Figure Legend Snippet: Analysis of the correlation between RUNX3 and DNMTs or EZH2 protein expression in AT2 cells. RUNX3 protein expression correlated positively with DNMT1 protein expression ( a ), but negatively with DNMT3b ( b ) and EZH2 ( c ) protein expression in AT2 cells. * P

    Techniques Used: Expressing

    DNMTs and EZH2 protein localization in lung tissues following hyperoxia. DNMTs and EZH2 protein localization in lung tissues by immunohistochemistry. After 10 day of hyperoxia, DNMT1 protein was expressed in pulmonary alveolar epithelial cells in the control group ( a ), but, in the model group, DNMT1 protein was barely detectable ( b ). In both the control ( c ) and model groups ( d ), DNMT3b protein was expressed in alveolar epithelial and mesenchymal cells. In the control group, EZH2 protein was poorly expressed ( e ). In the model group, EZH2 protein was expressed in alveolar epithelial cells ( f ). The arrow points to positive cells. (×400)
    Figure Legend Snippet: DNMTs and EZH2 protein localization in lung tissues following hyperoxia. DNMTs and EZH2 protein localization in lung tissues by immunohistochemistry. After 10 day of hyperoxia, DNMT1 protein was expressed in pulmonary alveolar epithelial cells in the control group ( a ), but, in the model group, DNMT1 protein was barely detectable ( b ). In both the control ( c ) and model groups ( d ), DNMT3b protein was expressed in alveolar epithelial and mesenchymal cells. In the control group, EZH2 protein was poorly expressed ( e ). In the model group, EZH2 protein was expressed in alveolar epithelial cells ( f ). The arrow points to positive cells. (×400)

    Techniques Used: Immunohistochemistry

    26) Product Images from "DNMT1 recruited by EZH2-mediated silencing of miR-484 contributes to the malignancy of cervical cancer cells through MMP14 and HNF1A"

    Article Title: DNMT1 recruited by EZH2-mediated silencing of miR-484 contributes to the malignancy of cervical cancer cells through MMP14 and HNF1A

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-019-0786-y

    miR-484 is repressed by hypermethylation mediated by EZH2-recruited DNMT1. a Schematic location of the core DNA motif (red) of the Polycomb response element (PRE). b , c PCR ( b ) and CHIP-qPCR ( c ) assay for anti-EZH2 and H3K27me3 in CC cells. d Western blot analysis of EZH2, H3K27me3, and Histone3 in CC cells with EZH2 overexpression or downregulation or control. e , f CHIP-qPCR assays for CC cells after EZH2 modification. g Physical interactions between EZH2 and DNMT1 were examined via a Co-IP assay in CC cells with EZH2 overexpression or downregulation. h CHIP-qPCR assays for anti-DNMT1 in CC cells after EZH2 downregulation. Mean ( n = 3) ± SD. Student’s t test, * p
    Figure Legend Snippet: miR-484 is repressed by hypermethylation mediated by EZH2-recruited DNMT1. a Schematic location of the core DNA motif (red) of the Polycomb response element (PRE). b , c PCR ( b ) and CHIP-qPCR ( c ) assay for anti-EZH2 and H3K27me3 in CC cells. d Western blot analysis of EZH2, H3K27me3, and Histone3 in CC cells with EZH2 overexpression or downregulation or control. e , f CHIP-qPCR assays for CC cells after EZH2 modification. g Physical interactions between EZH2 and DNMT1 were examined via a Co-IP assay in CC cells with EZH2 overexpression or downregulation. h CHIP-qPCR assays for anti-DNMT1 in CC cells after EZH2 downregulation. Mean ( n = 3) ± SD. Student’s t test, * p

    Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Modification, Co-Immunoprecipitation Assay

    27) Product Images from "Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth"

    Article Title: Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12800

    In vivo reactivation of miR‐326 and ARRB1 expression by EZH2 knockdown in MB CSCs. Xenograft tumors (XTs) generated in immunocompromised mice using D283 CSC transduced with lentiviral shEZH2 (XT‐shEZH2) or sh Scramble (XT‐Mock). (A) Left: Representative images of H E‐stained, largest‐diameter XT sections. Arrows indicate tumor masses. Magnification: 40× (left column), 400× (right). Scale bar, 100 µm. Right: Bar graphs showing XT volumes on postimplantation day 28. Bars represent means (SD), P = 0.0005. (B, C) XT‐shEZH2 and XT‐Mock expression of (B) (left) EZH2, ARRB1, E2F1‐ac and H3K27me3 proteins (ACTIN, GAPDH: loading control), (left) miR‐326 P = 0.0004, (C) mRNA for markers of differentiation (neuronal: βIIItub P
    Figure Legend Snippet: In vivo reactivation of miR‐326 and ARRB1 expression by EZH2 knockdown in MB CSCs. Xenograft tumors (XTs) generated in immunocompromised mice using D283 CSC transduced with lentiviral shEZH2 (XT‐shEZH2) or sh Scramble (XT‐Mock). (A) Left: Representative images of H E‐stained, largest‐diameter XT sections. Arrows indicate tumor masses. Magnification: 40× (left column), 400× (right). Scale bar, 100 µm. Right: Bar graphs showing XT volumes on postimplantation day 28. Bars represent means (SD), P = 0.0005. (B, C) XT‐shEZH2 and XT‐Mock expression of (B) (left) EZH2, ARRB1, E2F1‐ac and H3K27me3 proteins (ACTIN, GAPDH: loading control), (left) miR‐326 P = 0.0004, (C) mRNA for markers of differentiation (neuronal: βIIItub P

    Techniques Used: In Vivo, Expressing, Generated, Mouse Assay, Transduction, Staining

    EZH2‐dependent regulation of miR‐326 and ARRB1. (A) EZH2 expression mRNA (top) and protein (bottom) in NAC (control), MB CSC and D283 CSC (vs. NAC: MB CSC P
    Figure Legend Snippet: EZH2‐dependent regulation of miR‐326 and ARRB1. (A) EZH2 expression mRNA (top) and protein (bottom) in NAC (control), MB CSC and D283 CSC (vs. NAC: MB CSC P

    Techniques Used: Expressing

    Impact of EZH2—miR‐326/ARRB1—E2F1 axis in MB CSCs. In MB CSCs, the miR‐326 and ARRB1 transcription unit remains in a poised state: ready to be transcribed thanks to the presence of the permissive (H3K4me3) chromatin mark, but prevented from doing so by the persistence/predominance of the repressive (H3K27me3) chromatin mark, which is catalyzed by the histone methyltransferase EZH2. In this state, the cells express high levels of nonacetylated E2F1 transcription factor, which favors their self‐renewal and proliferation. Reversal of the H3K4me3: H3K27me3 ratio de‐represses miR‐326 and ARRB1 transcription. Restoration of miR‐326 expression reduces the E2F1 levels. Re‐expression of ARRB1, in complex with p300, acetylates E2F1 (E2F1‐Ac), thereby redirecting the transcription factor's activity toward pro‐apoptotic gene targets (e.g., TP73, CASP3, CASP7 ).
    Figure Legend Snippet: Impact of EZH2—miR‐326/ARRB1—E2F1 axis in MB CSCs. In MB CSCs, the miR‐326 and ARRB1 transcription unit remains in a poised state: ready to be transcribed thanks to the presence of the permissive (H3K4me3) chromatin mark, but prevented from doing so by the persistence/predominance of the repressive (H3K27me3) chromatin mark, which is catalyzed by the histone methyltransferase EZH2. In this state, the cells express high levels of nonacetylated E2F1 transcription factor, which favors their self‐renewal and proliferation. Reversal of the H3K4me3: H3K27me3 ratio de‐represses miR‐326 and ARRB1 transcription. Restoration of miR‐326 expression reduces the E2F1 levels. Re‐expression of ARRB1, in complex with p300, acetylates E2F1 (E2F1‐Ac), thereby redirecting the transcription factor's activity toward pro‐apoptotic gene targets (e.g., TP73, CASP3, CASP7 ).

    Techniques Used: Expressing, Activity Assay

    28) Product Images from "Long noncoding RNA MRCCAT1 promotes metastasis of clear cell renal cell carcinoma via inhibiting NPR3 and activating p38-MAPK signaling"

    Article Title: Long noncoding RNA MRCCAT1 promotes metastasis of clear cell renal cell carcinoma via inhibiting NPR3 and activating p38-MAPK signaling

    Journal: Molecular Cancer

    doi: 10.1186/s12943-017-0681-0

    MRCCAT1 suppresses NPR3 expression by recruiting Polycomb Repressive Complex 2 to NPR3 promoter. a qRT-PCR analysis of MRCCAT1 in subcellular fraction of Caki-1 cells. U6 and β-actin acted as nucleus and cytoplasm marker, respectively. b RIP assay analysis of the enrichment of MRCCAT1 to EZH2 in 786-O and Caki-1 cells. c Biotinylated MRCCAT1 or antisense RNA were incubated with nuclear extracts of 786-O and Caki-1 cells, targeted with streptavidin beads, and washed. Then the associated proteins were resolved in a gel. Western blot analysis of the specific association of EZH2 and MRCCAT1. d ChIP assays were conducted on NPR3 promoter regions using the indicated antibodies. Enrichment was determined relative to input controls. The results are presented as the mean ± SD from three independent experiments; ** P
    Figure Legend Snippet: MRCCAT1 suppresses NPR3 expression by recruiting Polycomb Repressive Complex 2 to NPR3 promoter. a qRT-PCR analysis of MRCCAT1 in subcellular fraction of Caki-1 cells. U6 and β-actin acted as nucleus and cytoplasm marker, respectively. b RIP assay analysis of the enrichment of MRCCAT1 to EZH2 in 786-O and Caki-1 cells. c Biotinylated MRCCAT1 or antisense RNA were incubated with nuclear extracts of 786-O and Caki-1 cells, targeted with streptavidin beads, and washed. Then the associated proteins were resolved in a gel. Western blot analysis of the specific association of EZH2 and MRCCAT1. d ChIP assays were conducted on NPR3 promoter regions using the indicated antibodies. Enrichment was determined relative to input controls. The results are presented as the mean ± SD from three independent experiments; ** P

    Techniques Used: Expressing, Quantitative RT-PCR, Marker, Incubation, Western Blot, Chromatin Immunoprecipitation

    29) Product Images from "Upregulation of lncRNA LINC00460 Facilitates GC Progression through Epigenetically Silencing CCNG2 by EZH2/LSD1 and Indicates Poor Outcomes"

    Article Title: Upregulation of lncRNA LINC00460 Facilitates GC Progression through Epigenetically Silencing CCNG2 by EZH2/LSD1 and Indicates Poor Outcomes

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.12.041

    Downstream Genes of LINC00460 and a Common Set of Target Genes Shared by LINC00460, EZH2, and LSD1 (A) Hierarchically clustered heatmap of the upregulated and downregulated genes in BGC823 cells after LINC00460 and NC siRNA transfections. (B) The scatterplot was used to assess the differences in gene expression between the GC cells transfected with LINC00460 and NC siRNAs. The values of x and y axis represented log 10 transformed gene expression level. Red color represented the increased genes, blue color represented the decreased genes, and gray color represented the genes with unchanged expression levels. (C) Pathway classification of differentially expressed genes (DEGs). x axis represented the number of DEGs, y axis represented the functional classification of KEGG. The change in gene mRNA levels was selectively verified using qRT-PCR after the knockout of LINC00460 both in BGC823 (D) and AGS (E) cells. (F) LINC00460 expression levels in GC nuclei or cytoplasm were detected by qRT-PCR. (G) FISH was conducted to measure the distribution of LINC00460 in GC cells (green, LINC00460; blue, DAPI). (H) RNA-protein interaction was performed to predict the interactions between LINC00460 and RBPs (probability > 0.5 was considered positive). (I) RIP was carried out, and the co-precipitated RNA was analyzed to determine LINC00460 (GAPDH as the internal control). (J) RNA pull-down assay was conducted. LINC00460 and antisense RNA were treated with cell extracts, and then EZH2 and LSD1 proteins were measured using western blotting (GAPDH as the internal control). (K) The mRNA levels of EZH2 and LSD1 in BGC823 and AGS cells were measured after LINC00460 knockout. The change in gene mRNA levels was selectively verified after EZH2 (L) or LSD1 (M) knockout. *p
    Figure Legend Snippet: Downstream Genes of LINC00460 and a Common Set of Target Genes Shared by LINC00460, EZH2, and LSD1 (A) Hierarchically clustered heatmap of the upregulated and downregulated genes in BGC823 cells after LINC00460 and NC siRNA transfections. (B) The scatterplot was used to assess the differences in gene expression between the GC cells transfected with LINC00460 and NC siRNAs. The values of x and y axis represented log 10 transformed gene expression level. Red color represented the increased genes, blue color represented the decreased genes, and gray color represented the genes with unchanged expression levels. (C) Pathway classification of differentially expressed genes (DEGs). x axis represented the number of DEGs, y axis represented the functional classification of KEGG. The change in gene mRNA levels was selectively verified using qRT-PCR after the knockout of LINC00460 both in BGC823 (D) and AGS (E) cells. (F) LINC00460 expression levels in GC nuclei or cytoplasm were detected by qRT-PCR. (G) FISH was conducted to measure the distribution of LINC00460 in GC cells (green, LINC00460; blue, DAPI). (H) RNA-protein interaction was performed to predict the interactions between LINC00460 and RBPs (probability > 0.5 was considered positive). (I) RIP was carried out, and the co-precipitated RNA was analyzed to determine LINC00460 (GAPDH as the internal control). (J) RNA pull-down assay was conducted. LINC00460 and antisense RNA were treated with cell extracts, and then EZH2 and LSD1 proteins were measured using western blotting (GAPDH as the internal control). (K) The mRNA levels of EZH2 and LSD1 in BGC823 and AGS cells were measured after LINC00460 knockout. The change in gene mRNA levels was selectively verified after EZH2 (L) or LSD1 (M) knockout. *p

    Techniques Used: Transfection, Expressing, Transformation Assay, Functional Assay, Quantitative RT-PCR, Knock-Out, Fluorescence In Situ Hybridization, Pull Down Assay, Western Blot

    LINC00460 Regulates the Transcriptions of Multiple Genes through Interacting with EZH2 and LSD1 (A) Seven tumor suppressor genes were simultaneously changed in the GC cells transfected with si-LINC00460, si-EZH2, or si-LSD1. ChIP-qPCR of EZH2/H3K27me3 and LSD1/H3K4me2 in gene promoter regions (including CCNG2 (B), IFI44 (C), IFIT2(D), P21 (E), Bax (F), KLF2 (G)) after the transfections with NC or LINC00460 siRNAs in BGC823 cells. Enrichment was quantified with the anti-IgG antibody as an internal control. (H) Analysis for the differentially expressed genes between GC and non-tumor samples in GSE54129 dataset. Data were expressed as mean ± SD of three independent trials. *p
    Figure Legend Snippet: LINC00460 Regulates the Transcriptions of Multiple Genes through Interacting with EZH2 and LSD1 (A) Seven tumor suppressor genes were simultaneously changed in the GC cells transfected with si-LINC00460, si-EZH2, or si-LSD1. ChIP-qPCR of EZH2/H3K27me3 and LSD1/H3K4me2 in gene promoter regions (including CCNG2 (B), IFI44 (C), IFIT2(D), P21 (E), Bax (F), KLF2 (G)) after the transfections with NC or LINC00460 siRNAs in BGC823 cells. Enrichment was quantified with the anti-IgG antibody as an internal control. (H) Analysis for the differentially expressed genes between GC and non-tumor samples in GSE54129 dataset. Data were expressed as mean ± SD of three independent trials. *p

    Techniques Used: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    30) Product Images from "Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes"

    Article Title: Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky595

    Enrichment of H3 marks and PRC2 at the endogenous loci of PRE-like DNA fragments. ( A ) ChIP-qPCR of H3 (open bars), H3K27me3 (dotted open bars) and H3K4me3 (filled solid bars) at the endogenous loci of PRE-like DNA fragments in HeLa cells shown as % input. ( B ) ChIP-qPCR of EZH2 (open bars) and EED (filled solid bars) shown as % input. ( C ) The expression of SUZ12 in SUZ12 shRNA knockdown cell line was significantly reduced as compared to that in control cell line treated with scrambled shRNA. ( D ) The luciferase activity of PRE-like DNA fragments in SUZ12 knockdown cells (filled solid bars) compared to control cell line treated with scrambled shRNA (open bars). The RLU at Gene Desert was considered as 100%. ( E ) ChIP-qPCR of EED at the endogenous loci of PRE-like DNA fragments in SUZ12 knockdown cell line shown as % input. In a, b, d, e , data were represented as mean ± SD from at least three biological replicates that were performed on different samples on different days. The −1, −2 labels in a, b, e . The P values of Student's t -test comparing SUZ12 shRNA and scrambled shRNA ( D, E ) were showed as * P
    Figure Legend Snippet: Enrichment of H3 marks and PRC2 at the endogenous loci of PRE-like DNA fragments. ( A ) ChIP-qPCR of H3 (open bars), H3K27me3 (dotted open bars) and H3K4me3 (filled solid bars) at the endogenous loci of PRE-like DNA fragments in HeLa cells shown as % input. ( B ) ChIP-qPCR of EZH2 (open bars) and EED (filled solid bars) shown as % input. ( C ) The expression of SUZ12 in SUZ12 shRNA knockdown cell line was significantly reduced as compared to that in control cell line treated with scrambled shRNA. ( D ) The luciferase activity of PRE-like DNA fragments in SUZ12 knockdown cells (filled solid bars) compared to control cell line treated with scrambled shRNA (open bars). The RLU at Gene Desert was considered as 100%. ( E ) ChIP-qPCR of EED at the endogenous loci of PRE-like DNA fragments in SUZ12 knockdown cell line shown as % input. In a, b, d, e , data were represented as mean ± SD from at least three biological replicates that were performed on different samples on different days. The −1, −2 labels in a, b, e . The P values of Student's t -test comparing SUZ12 shRNA and scrambled shRNA ( D, E ) were showed as * P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, shRNA, Luciferase, Activity Assay

    31) Product Images from "KDM2B is involved in the epigenetic regulation of TGF-β-induced epithelial–mesenchymal transition in lung and pancreatic cancer cell lines"

    Article Title: KDM2B is involved in the epigenetic regulation of TGF-β-induced epithelial–mesenchymal transition in lung and pancreatic cancer cell lines

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.015502

    KDM2B influenced the regulation of histone H2A ubiquitination and H3 methylation on the regulatory regions of several epithelial marker genes in A549 cells. A549 cells were infected with the control retrovirus or the retrovirus expressing wild-type KDM2B (WT) with or without TGF-β treatment. ChIP analyses of H2AK119Ub, KDM2B, H3K27me3, and EZH2 on the regulatory regions of CDH1 ( A ), miR200a/b ( B ), CGN ( C ), and GAPDH genes ( D ) in A549 cells are shown. The occupancies of ubiquitinated histones, KDM2B, methylated histones or EZH2 proteins on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (n = 3) (∗∗ p
    Figure Legend Snippet: KDM2B influenced the regulation of histone H2A ubiquitination and H3 methylation on the regulatory regions of several epithelial marker genes in A549 cells. A549 cells were infected with the control retrovirus or the retrovirus expressing wild-type KDM2B (WT) with or without TGF-β treatment. ChIP analyses of H2AK119Ub, KDM2B, H3K27me3, and EZH2 on the regulatory regions of CDH1 ( A ), miR200a/b ( B ), CGN ( C ), and GAPDH genes ( D ) in A549 cells are shown. The occupancies of ubiquitinated histones, KDM2B, methylated histones or EZH2 proteins on the regions were analyzed by quantitative PCR. Percentage enrichment over input chromatin DNA was presented (n = 3) (∗∗ p

    Techniques Used: Methylation, Marker, Infection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    32) Product Images from "Comprehensive Evaluation of the Role of EZH2 in the Growth, Invasion, and Aggression of a Panel of Prostate Cancer Cell Lines"

    Article Title: Comprehensive Evaluation of the Role of EZH2 in the Growth, Invasion, and Aggression of a Panel of Prostate Cancer Cell Lines

    Journal: The Prostate

    doi: 10.1002/pros.21112

    Immunocytochemistry confirms functionality of overexpressed EZH2 and of shEZH2
    Figure Legend Snippet: Immunocytochemistry confirms functionality of overexpressed EZH2 and of shEZH2

    Techniques Used: Immunocytochemistry

    EZH2 modifies phenotype of all prostate cancer cell lines upon knockdown and overexpression
    Figure Legend Snippet: EZH2 modifies phenotype of all prostate cancer cell lines upon knockdown and overexpression

    Techniques Used: Over Expression

    Expression levels of EZH2 after transduction with overexpression and shRNA viruses
    Figure Legend Snippet: Expression levels of EZH2 after transduction with overexpression and shRNA viruses

    Techniques Used: Expressing, Transduction, Over Expression, shRNA

    EZH2-modulation alters colony growth of some, but not all prostate cancer cell lines
    Figure Legend Snippet: EZH2-modulation alters colony growth of some, but not all prostate cancer cell lines

    Techniques Used:

    Altered expression of EZH2 impacts the invasive behavior of prostate cancer cell lines to varying degrees
    Figure Legend Snippet: Altered expression of EZH2 impacts the invasive behavior of prostate cancer cell lines to varying degrees

    Techniques Used: Expressing

    EZH2 overexpression and knockdown phenotypes extend to in vivo tumor growth
    Figure Legend Snippet: EZH2 overexpression and knockdown phenotypes extend to in vivo tumor growth

    Techniques Used: Over Expression, In Vivo

    EZH2 knockdown decreases cellular growth rate, while EZH2 overexpression has no appreciable effect
    Figure Legend Snippet: EZH2 knockdown decreases cellular growth rate, while EZH2 overexpression has no appreciable effect

    Techniques Used: Over Expression

    EZH2 expression, overexpression, and knockdown in prostate cancer cell lines
    Figure Legend Snippet: EZH2 expression, overexpression, and knockdown in prostate cancer cell lines

    Techniques Used: Expressing, Over Expression

    33) Product Images from "Sodium Arsenite Represses the Expression of Myogenin in C2C12 Mouse Myoblast Cells Through Histone Modifications and Altered Expression of Ezh2, Glp, and Igf-1"

    Article Title: Sodium Arsenite Represses the Expression of Myogenin in C2C12 Mouse Myoblast Cells Through Histone Modifications and Altered Expression of Ezh2, Glp, and Igf-1

    Journal: Toxicology and Applied Pharmacology

    doi: 10.1016/j.taap.2012.03.002

    Arsenic exposure enhances Ezh2 nuclear expression and recruits Ezh2 to the myogenin promoter near the transcription start site
    Figure Legend Snippet: Arsenic exposure enhances Ezh2 nuclear expression and recruits Ezh2 to the myogenin promoter near the transcription start site

    Techniques Used: Expressing

    Ezh2 recruits Dnmt 3a, but not Dnmt 3b, to the myogenin promoter
    Figure Legend Snippet: Ezh2 recruits Dnmt 3a, but not Dnmt 3b, to the myogenin promoter

    Techniques Used:

    34) Product Images from "LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells"

    Article Title: LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171070

    DANCR epigenetically suppresses lncRNA-LET expression through association with EZH2 and HDAC3 ( A ) The DANCR-overexpressed AGS cells were treated with 5 μM DZNep and/or 1 μM SAHA for 48 h, and the relative expression of lncRNA-LET was detected by qPCR. ( B ) The DANCR-overexpressed AGS cells were transfected with EZH2 and/or HDAC3. After 48 h, the relative expression of lncRNA-LET was detected by qPCR. ( C ) DANCR RNA levels in immunoprecipitates by EZH2 or HDAC3 were determined by qPCR. DANCR RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. ( D ) EZH2 and HDAC3 protein levels in immunoprecipitates with biotin-labeled DANCR RNA were evaluated by Western blot. ( E ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-silenced BCG-823 cells. ( F ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-overexpressed AGS cells. ( G ) BCG-823 cells were transfected with DANCR siRNAs for 48 h. After immunoprecipitating endogenous EZH2, bound HDAC3 was subjected to Western blotting. All experiments were repeated three times. Data are shown as mean ± SD; * P
    Figure Legend Snippet: DANCR epigenetically suppresses lncRNA-LET expression through association with EZH2 and HDAC3 ( A ) The DANCR-overexpressed AGS cells were treated with 5 μM DZNep and/or 1 μM SAHA for 48 h, and the relative expression of lncRNA-LET was detected by qPCR. ( B ) The DANCR-overexpressed AGS cells were transfected with EZH2 and/or HDAC3. After 48 h, the relative expression of lncRNA-LET was detected by qPCR. ( C ) DANCR RNA levels in immunoprecipitates by EZH2 or HDAC3 were determined by qPCR. DANCR RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. ( D ) EZH2 and HDAC3 protein levels in immunoprecipitates with biotin-labeled DANCR RNA were evaluated by Western blot. ( E ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-silenced BCG-823 cells. ( F ) The occupancy level of EZH2, HDAC3, H3K27me3, H3Ac, and H4Ac at lncRNA-LET promoter region was determined by ChIP assay and followed by qPCR in control and DANCR-overexpressed AGS cells. ( G ) BCG-823 cells were transfected with DANCR siRNAs for 48 h. After immunoprecipitating endogenous EZH2, bound HDAC3 was subjected to Western blotting. All experiments were repeated three times. Data are shown as mean ± SD; * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, RNA Expression, Labeling, Western Blot, Chromatin Immunoprecipitation

    35) Product Images from "Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity"

    Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

    Journal: eLife

    doi: 10.7554/eLife.38080

    Characterization of Meg3 isoforms ( A ) Upper panel: Time-series expression of the PRC2 subunits ( Eed , Suz12 , Jarid2 , and Ezh2 ) during ESC~MN differentiation. The levels of PRC2 complex progressively decreased during differentiation, whereas Jarid2 is reactivated in day 7 postmitotic MNs. Lower panel: The abundances of the known lncRNAs that interact with PRC2 in MNs, as revealed by RNA-seq. ( B ) RNA-seq analysis of ESC~MNs. Reads from ESCs, RA-induced nascent neural epithelium (NE at day 2), MN progenitors (pMN at day 4), postmitotic interneurons (IN at day 7), and postmitotic MNs (MN at day 7). Reads are normalized to the total number of mappable reads per sample. RNA-seq revealed that Meg3 v1 (blue boxes) and Meg3 v5 (pink boxes) are the most abundant Meg3 isoforms in postmitotic MNs (GENCODE version M9). ( C ) Radar chart reveals that day 7 postmitotic MNs have the highest distribution of Meg3 v5 during ESC~MN differentiation. ( D ) Histogram plot indicates that the Meg3 v1 and Meg3 v5 isoforms account for more than 99% of Meg3 transcripts during ESC~MN differentiation. ( E ) Schematic diagram of RT-PCR primer locations within the Meg3 v1 and Meg3 v5 regions. Expression of Meg3 according to different primer combinations for Meg3 isoforms suggests that Meg3 v1 and Meg3 v5 are independent transcripts. Gapdh as a loading control. ( F ) Western blot shows that the loss of Meg3 imprinted lncRNAs does not affect the protein abundance of Ezh2 and Jarid2 in ESC~MNs.
    Figure Legend Snippet: Characterization of Meg3 isoforms ( A ) Upper panel: Time-series expression of the PRC2 subunits ( Eed , Suz12 , Jarid2 , and Ezh2 ) during ESC~MN differentiation. The levels of PRC2 complex progressively decreased during differentiation, whereas Jarid2 is reactivated in day 7 postmitotic MNs. Lower panel: The abundances of the known lncRNAs that interact with PRC2 in MNs, as revealed by RNA-seq. ( B ) RNA-seq analysis of ESC~MNs. Reads from ESCs, RA-induced nascent neural epithelium (NE at day 2), MN progenitors (pMN at day 4), postmitotic interneurons (IN at day 7), and postmitotic MNs (MN at day 7). Reads are normalized to the total number of mappable reads per sample. RNA-seq revealed that Meg3 v1 (blue boxes) and Meg3 v5 (pink boxes) are the most abundant Meg3 isoforms in postmitotic MNs (GENCODE version M9). ( C ) Radar chart reveals that day 7 postmitotic MNs have the highest distribution of Meg3 v5 during ESC~MN differentiation. ( D ) Histogram plot indicates that the Meg3 v1 and Meg3 v5 isoforms account for more than 99% of Meg3 transcripts during ESC~MN differentiation. ( E ) Schematic diagram of RT-PCR primer locations within the Meg3 v1 and Meg3 v5 regions. Expression of Meg3 according to different primer combinations for Meg3 isoforms suggests that Meg3 v1 and Meg3 v5 are independent transcripts. Gapdh as a loading control. ( F ) Western blot shows that the loss of Meg3 imprinted lncRNAs does not affect the protein abundance of Ezh2 and Jarid2 in ESC~MNs.

    Techniques Used: Expressing, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Ezh2 binding and the H3K27me3 landscape in ESC~MNs ( A and B ) Concordant enrichments of Ezh2 and H3K27me3 in progenitor genes ( A ) and caudal Hox genes ( B ).(C~E) ChIP-qPCR verifies compromised H3K27me3 occupancy of Ezh2 and Jarid2 for the Pax6 , Irx3 , and Hoxc8 loci upon Meg3 KD. 1% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments; * p-value
    Figure Legend Snippet: Ezh2 binding and the H3K27me3 landscape in ESC~MNs ( A and B ) Concordant enrichments of Ezh2 and H3K27me3 in progenitor genes ( A ) and caudal Hox genes ( B ).(C~E) ChIP-qPCR verifies compromised H3K27me3 occupancy of Ezh2 and Jarid2 for the Pax6 , Irx3 , and Hoxc8 loci upon Meg3 KD. 1% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments; * p-value

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    36) Product Images from "LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma, et al. LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma"

    Article Title: LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma, et al. LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.16480

    LncRNA is responsible for the epigenetic repression of YAP1 by interacting with PRC2. A, The PWAR6 subcellular location was determined in AsPC‐1 and BxPC‐3 cells. U6 was used as a nuclear marker and GADPH was used as a cytoplasmic marker. B, The binding probability between PWAR6 and PRC2 complex (SUZ12, EZH2, EED) was predicted in RNA‐protein interaction prediction website (RF and SVM scores > 0.5). C, RIP assay validated the interaction between PWAR6 and EZH2/SUZ12 in PDAC cells. D, RNA pull‐down assay demonstrated that PWAR6 could specially retrieved EZH2 in PDAC cells. E, The results of ChIP assay shown that PWAR6 overexpression could dramatically enhancing the binding ability of EZH2 to YAP1 promoter region and promoting the methylation of H3K27me3 in PDAC cells, while PWAR6 down‐regulation contributed to the contrary results
    Figure Legend Snippet: LncRNA is responsible for the epigenetic repression of YAP1 by interacting with PRC2. A, The PWAR6 subcellular location was determined in AsPC‐1 and BxPC‐3 cells. U6 was used as a nuclear marker and GADPH was used as a cytoplasmic marker. B, The binding probability between PWAR6 and PRC2 complex (SUZ12, EZH2, EED) was predicted in RNA‐protein interaction prediction website (RF and SVM scores > 0.5). C, RIP assay validated the interaction between PWAR6 and EZH2/SUZ12 in PDAC cells. D, RNA pull‐down assay demonstrated that PWAR6 could specially retrieved EZH2 in PDAC cells. E, The results of ChIP assay shown that PWAR6 overexpression could dramatically enhancing the binding ability of EZH2 to YAP1 promoter region and promoting the methylation of H3K27me3 in PDAC cells, while PWAR6 down‐regulation contributed to the contrary results

    Techniques Used: Marker, Binding Assay, Pull Down Assay, Chromatin Immunoprecipitation, Over Expression, Methylation

    37) Product Images from "SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer"

    Article Title: SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer

    Journal: Oncogene

    doi: 10.1038/onc.2016.300

    Skp2 deficiency decreases the levels of Ezh2 and H3K27me3 in vitro and in vivo . ( a ) Western blot analysis of protein levels of Ezh2 and H3K27me3 in Pten/Trp53 double-null and Pten/Trp53/Skp2 triple-null MEFs. ( b ) Quantification analysis of protein levels for Ezh2 and H3K27me3 in MEFs upon Skp2 inactivation from ( a ). Error bars represent means ±s.d. ( c ) IHC staining of Ezh2 and H3K27me3 in prostate tumors of Pten/ Trp53 and Pten/Trp53/Skp2 mutant mice ( n = 3) at 4 months of age. Scale bars represent 50 μm (inset 100 μm). ( d ) Quantification analysis of tumor cells positive for Ezh2 and H3K27me3 from ( c ). Error bars represent means ±s.d. from three mice for each group.
    Figure Legend Snippet: Skp2 deficiency decreases the levels of Ezh2 and H3K27me3 in vitro and in vivo . ( a ) Western blot analysis of protein levels of Ezh2 and H3K27me3 in Pten/Trp53 double-null and Pten/Trp53/Skp2 triple-null MEFs. ( b ) Quantification analysis of protein levels for Ezh2 and H3K27me3 in MEFs upon Skp2 inactivation from ( a ). Error bars represent means ±s.d. ( c ) IHC staining of Ezh2 and H3K27me3 in prostate tumors of Pten/ Trp53 and Pten/Trp53/Skp2 mutant mice ( n = 3) at 4 months of age. Scale bars represent 50 μm (inset 100 μm). ( d ) Quantification analysis of tumor cells positive for Ezh2 and H3K27me3 from ( c ). Error bars represent means ±s.d. from three mice for each group.

    Techniques Used: In Vitro, In Vivo, Western Blot, Immunohistochemistry, Staining, Mutagenesis, Mouse Assay

    Skp2 and Ezh2 are increased in recurrent prostate tumors of Pten/Trp53 mice, and SKP2 is correlated with EZH2 in human prostate cancer. ( a ) IHC staining of Skp2, Ezh2, H3K27me3 and Ki67 in regressive and recurrent lesions of prostate tumors of castrated Luc/Pten/Trp53 ( b ) Quantification analysis of protein levels for Skp2, Ezh2, H3K27me3 and Ki67 in prostate tumors from ( a ). ( c ) IHC staining on SKP2 and EZH2 in human PCa tissue microarray (TMA). Scale bars represent 50 μm (inset 100 μm) in ( a and c ). ( d ). ( e ) A working model of SKP2, EZH2 and TRAF6 network on the epigenetic regulation of H3K27me3 in PCa. SKP2 stabilizes EZH2 by a sequestration of TRAF6-mediated ubiquitination for EZH2 degradation, and SKP2 deficiency results in a reduction of EZH2 and H3K27me3 to suppress PCa progression.
    Figure Legend Snippet: Skp2 and Ezh2 are increased in recurrent prostate tumors of Pten/Trp53 mice, and SKP2 is correlated with EZH2 in human prostate cancer. ( a ) IHC staining of Skp2, Ezh2, H3K27me3 and Ki67 in regressive and recurrent lesions of prostate tumors of castrated Luc/Pten/Trp53 ( b ) Quantification analysis of protein levels for Skp2, Ezh2, H3K27me3 and Ki67 in prostate tumors from ( a ). ( c ) IHC staining on SKP2 and EZH2 in human PCa tissue microarray (TMA). Scale bars represent 50 μm (inset 100 μm) in ( a and c ). ( d ). ( e ) A working model of SKP2, EZH2 and TRAF6 network on the epigenetic regulation of H3K27me3 in PCa. SKP2 stabilizes EZH2 by a sequestration of TRAF6-mediated ubiquitination for EZH2 degradation, and SKP2 deficiency results in a reduction of EZH2 and H3K27me3 to suppress PCa progression.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Microarray

    Aberrant elevation of Skp2 and Ezh2 upon Pten loss in vitro in MEFs and in vivo in mouse tissues. ( a ) Top panel: Western blot analysis to show the increases of protein levels of Skp2, Ezh2 and H3K27me3 upon Pten loss in MEFs. Bottom panel: Quantification analysis of protein levels for Skp2, Ezh2 and H3K27me3 from ( a ). Error bars represent means ±s.d. ( P
    Figure Legend Snippet: Aberrant elevation of Skp2 and Ezh2 upon Pten loss in vitro in MEFs and in vivo in mouse tissues. ( a ) Top panel: Western blot analysis to show the increases of protein levels of Skp2, Ezh2 and H3K27me3 upon Pten loss in MEFs. Bottom panel: Quantification analysis of protein levels for Skp2, Ezh2 and H3K27me3 from ( a ). Error bars represent means ±s.d. ( P

    Techniques Used: In Vitro, In Vivo, Western Blot

    EZH2 stability is co-regulated by SKP2 and TRAF6 in human prostate cancer cells. ( a ) Western blot analysis of EZH2 and H3K27me3 in PC3-scrambled and PC3-shSKP2 PCa cells. ( b ) Quantitative RT-qPCR analysis of the mRNA levels of EZH2 in PC3 cells upon SKP2 knockdown. Error bars represent means ±s.d. of triplicates. ( c ) SKP2 knockdown shortens the half-life of EZH2. Top panel: Western blot analysis of the half-life of EZH2 upon SKP2 knockdown in PC3 cells. Cells were treated with cycloheximide (CHX, 100 μg/ml), and then levels of EZH2 were detected at defined time points. Bottom panel: Quantification of EZH2 normalized to β-actin from the top panel. ( d ) SKP2 overexpression inhibits TRAF6 but upregulates EZH2 in C4-2B cells. EV, Empty vector. ( e ) TRAF6 knockdown upregulates EZH2 and H3K27me3 in PC3 cells. Two different sets of siTRAF6 oligos were used. ( f ) TRAF6 overexpression decreases EZH2 and H3K27me3 in PC3 cells. ( g ) Co-immunoprecipitation analysis indicates a physical interaction between endogenous EZH2 and TRAF6 proteins in PC3 cells. ( h ) Immunofluorescence images show a co-localization of endogenous EZH2 and TRAF6 in PC3 cells. Scale bar represents 5 μm.
    Figure Legend Snippet: EZH2 stability is co-regulated by SKP2 and TRAF6 in human prostate cancer cells. ( a ) Western blot analysis of EZH2 and H3K27me3 in PC3-scrambled and PC3-shSKP2 PCa cells. ( b ) Quantitative RT-qPCR analysis of the mRNA levels of EZH2 in PC3 cells upon SKP2 knockdown. Error bars represent means ±s.d. of triplicates. ( c ) SKP2 knockdown shortens the half-life of EZH2. Top panel: Western blot analysis of the half-life of EZH2 upon SKP2 knockdown in PC3 cells. Cells were treated with cycloheximide (CHX, 100 μg/ml), and then levels of EZH2 were detected at defined time points. Bottom panel: Quantification of EZH2 normalized to β-actin from the top panel. ( d ) SKP2 overexpression inhibits TRAF6 but upregulates EZH2 in C4-2B cells. EV, Empty vector. ( e ) TRAF6 knockdown upregulates EZH2 and H3K27me3 in PC3 cells. Two different sets of siTRAF6 oligos were used. ( f ) TRAF6 overexpression decreases EZH2 and H3K27me3 in PC3 cells. ( g ) Co-immunoprecipitation analysis indicates a physical interaction between endogenous EZH2 and TRAF6 proteins in PC3 cells. ( h ) Immunofluorescence images show a co-localization of endogenous EZH2 and TRAF6 in PC3 cells. Scale bar represents 5 μm.

    Techniques Used: Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Immunoprecipitation, Immunofluorescence

    TRAF6 is an E3 ubiquitin ligase for EZH2 ubiquitination through lysine 63-linkage. ( a ) In vivo ubiquitination assay showed that TRAF6 promotes the K63-linked polyubiquitination of EZH2 in HEK293T cells. Cells were transfected with Flag-EZH2, Myc-TRAF6, along with various HA-ubiquitin (HA-Ub) constructs. K48 and K63 represent HA-Ub-K48-only and HA-Ub-K63-only, respectively. WCL represents whole cell lysates. ( b ) Endogenous IP assay demonstrated that TRAF6 knockdown results in a reduction of EZH2 polyubiquitination in PC3 cells. ( c ) TRAF6 overexpression increases EZH2 polyubiquitination in C4-2B cells. ( d ) TRAF6 mutation at C70A dramatically decreases the TRAF6-mediated polyubiquitination of EZH2 in vivo . HEK293T cells were transfected with Flag-EZH2, HA-Ub-K63 only, along with TRAF6 WT or TRAF6 C70A (catalytically dead mutant). ( e ) TRAF6 mutation at C70A abolishes the TRAF6-mediated polyubiquitination of EZH2 in vitro . Flag-EZH2 proteins were incubated with adenosine triphosphate, HA-Ub, E1, and E2 (Ubc13/Uve1a), along with GST, GST-TRAF6, or GST-TRAF6-C70A proteins for in vitro ubiquitination of EZH2.
    Figure Legend Snippet: TRAF6 is an E3 ubiquitin ligase for EZH2 ubiquitination through lysine 63-linkage. ( a ) In vivo ubiquitination assay showed that TRAF6 promotes the K63-linked polyubiquitination of EZH2 in HEK293T cells. Cells were transfected with Flag-EZH2, Myc-TRAF6, along with various HA-ubiquitin (HA-Ub) constructs. K48 and K63 represent HA-Ub-K48-only and HA-Ub-K63-only, respectively. WCL represents whole cell lysates. ( b ) Endogenous IP assay demonstrated that TRAF6 knockdown results in a reduction of EZH2 polyubiquitination in PC3 cells. ( c ) TRAF6 overexpression increases EZH2 polyubiquitination in C4-2B cells. ( d ) TRAF6 mutation at C70A dramatically decreases the TRAF6-mediated polyubiquitination of EZH2 in vivo . HEK293T cells were transfected with Flag-EZH2, HA-Ub-K63 only, along with TRAF6 WT or TRAF6 C70A (catalytically dead mutant). ( e ) TRAF6 mutation at C70A abolishes the TRAF6-mediated polyubiquitination of EZH2 in vitro . Flag-EZH2 proteins were incubated with adenosine triphosphate, HA-Ub, E1, and E2 (Ubc13/Uve1a), along with GST, GST-TRAF6, or GST-TRAF6-C70A proteins for in vitro ubiquitination of EZH2.

    Techniques Used: In Vivo, Ubiquitin Assay, Transfection, Construct, Over Expression, Mutagenesis, In Vitro, Incubation

    38) Product Images from "Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models"

    Article Title: Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models

    Journal: Redox Biology

    doi: 10.1016/j.redox.2020.101839

    PSELT regulates EZH2/H3K27me3 levels to promote cell survival. A: SH-SY5Y cells were incubated with PSELT-dansyl for 15 min or 6 h and immunolabeled for EZH2. The nuclei were labeled by the TO-PRO dye. Scale bars: 10 and 20 μm B: Quantification of EZH2 immunoreactivity after exposure to PSELT-dansyl. Data are expressed as mean ± SEM and are compared using Student t -test, *** p
    Figure Legend Snippet: PSELT regulates EZH2/H3K27me3 levels to promote cell survival. A: SH-SY5Y cells were incubated with PSELT-dansyl for 15 min or 6 h and immunolabeled for EZH2. The nuclei were labeled by the TO-PRO dye. Scale bars: 10 and 20 μm B: Quantification of EZH2 immunoreactivity after exposure to PSELT-dansyl. Data are expressed as mean ± SEM and are compared using Student t -test, *** p

    Techniques Used: Incubation, Immunolabeling, Labeling

    PSELT effect on gene expression levels in SH-SY5Y cells correlates with different gene sets. A: Unsupervised clustering based on gene expression profiling of the different experimental conditions indicated under the dendrogram ( n = 3 per condition). Genes with a relatively higher level of expression are shown in red, and those with a lower level are shown in blue according to the color scale at the bottom. B: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated in Parkinson's disease ( p value = 0, enrichment score = 1.15). Genes up-regulated in Parkinson's disease positively correlate with genes up-regulated by MPP + and down-regulated by PSELT. Data were taken from GSE68719_Parkinson disease [ 36 ] or from GSE78757_Gaucher disease, GSE20292_Parkinson substantia nigra and GSE9397_Parkinson MSN [ [36] , [61] , [62] ] in supplementary Fig.S1A . C: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those down-regulated in cortical neurogenesis ( p value = 0, enrichment score = −1.29). Genes down-regulated during impaired corticogenesis correlate with genes up-regulated by PSELT. Data were taken from GSE74683_Cortex_Neurogenesis (ELP3 knockout) [ 63 ] or from GSE 42904_HIPPO [ 64 ], GSE8425_RASGRF1 KO [ 65 ] and the orphan nuclear receptor TLX targets [ 66 ] in supplementary Fig.S1B . D: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated during caloric restriction ( p value = 0, FDR = 0, enrichment score = 1.30). Genes that are up-regulated by caloric restriction are also up-regulated by PSELT. Data were taken from GSE75569 [ 67 ]. E: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated by resveratrol ( p value = 0, enrichment score = 1.23). Genes that are up-regulated by resveratrol are also up-regulated by PSELT and inversely, genes down-regulated by resveratrol are also down-regulated by PSELT. Data were taken from GSE36930 [ 68 ] or from GSE36930 [ 68 ] and GSE42432 [ 69 ] in supplementary Fig. S1C . F: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those associated/interacting with the transcription regulator EZH2 ( p value = 0, enrichment score = −1.25). The EZH2-interacting genes are mostly correlated with PSELT treatment. Data were taken from the NCBI database ( https://www.ncbi.nlm.nih.gov ). G: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated by EZH2 knockout ( p value = 0, enrichment score = 1.14). Genes that are up-regulated by EZH2 knockout and have a H3K27me3 mark correlate with genes up-regulated by MPP + and are down-regulated by PSELT. Data were taken from GSE80222 [ 38 ] and from several other studies [ [70] , [71] , [72] ] in supplementary Fig. S1D . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: PSELT effect on gene expression levels in SH-SY5Y cells correlates with different gene sets. A: Unsupervised clustering based on gene expression profiling of the different experimental conditions indicated under the dendrogram ( n = 3 per condition). Genes with a relatively higher level of expression are shown in red, and those with a lower level are shown in blue according to the color scale at the bottom. B: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated in Parkinson's disease ( p value = 0, enrichment score = 1.15). Genes up-regulated in Parkinson's disease positively correlate with genes up-regulated by MPP + and down-regulated by PSELT. Data were taken from GSE68719_Parkinson disease [ 36 ] or from GSE78757_Gaucher disease, GSE20292_Parkinson substantia nigra and GSE9397_Parkinson MSN [ [36] , [61] , [62] ] in supplementary Fig.S1A . C: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those down-regulated in cortical neurogenesis ( p value = 0, enrichment score = −1.29). Genes down-regulated during impaired corticogenesis correlate with genes up-regulated by PSELT. Data were taken from GSE74683_Cortex_Neurogenesis (ELP3 knockout) [ 63 ] or from GSE 42904_HIPPO [ 64 ], GSE8425_RASGRF1 KO [ 65 ] and the orphan nuclear receptor TLX targets [ 66 ] in supplementary Fig.S1B . D: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated during caloric restriction ( p value = 0, FDR = 0, enrichment score = 1.30). Genes that are up-regulated by caloric restriction are also up-regulated by PSELT. Data were taken from GSE75569 [ 67 ]. E: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated by resveratrol ( p value = 0, enrichment score = 1.23). Genes that are up-regulated by resveratrol are also up-regulated by PSELT and inversely, genes down-regulated by resveratrol are also down-regulated by PSELT. Data were taken from GSE36930 [ 68 ] or from GSE36930 [ 68 ] and GSE42432 [ 69 ] in supplementary Fig. S1C . F: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those associated/interacting with the transcription regulator EZH2 ( p value = 0, enrichment score = −1.25). The EZH2-interacting genes are mostly correlated with PSELT treatment. Data were taken from the NCBI database ( https://www.ncbi.nlm.nih.gov ). G: Gene set enrichment analysis of a ranked list of all genes comparing MPP + and PSELT treatment versus MPP + with those up-regulated by EZH2 knockout ( p value = 0, enrichment score = 1.14). Genes that are up-regulated by EZH2 knockout and have a H3K27me3 mark correlate with genes up-regulated by MPP + and are down-regulated by PSELT. Data were taken from GSE80222 [ 38 ] and from several other studies [ [70] , [71] , [72] ] in supplementary Fig. S1D . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Expressing, Knock-Out

    39) Product Images from "Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency"

    Article Title: Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency

    Journal: mBio

    doi: 10.1128/mBio.00133-17

    Distribution of histone methyltransferases and methylated histones on HIV proviruses following CRISPR disruption. (A) EZH2 and EHMT2 disruption. (B) JARID2 disruption. ChIP assays were performed with E4 cells infected with the CRISPRs indicated by using primers to the HIV LTR and downstream regions ( 27 ). HIV DNA levels were calculated as percentages of the input. Error bars represent the SEM of three separate real-time PCR measurements.
    Figure Legend Snippet: Distribution of histone methyltransferases and methylated histones on HIV proviruses following CRISPR disruption. (A) EZH2 and EHMT2 disruption. (B) JARID2 disruption. ChIP assays were performed with E4 cells infected with the CRISPRs indicated by using primers to the HIV LTR and downstream regions ( 27 ). HIV DNA levels were calculated as percentages of the input. Error bars represent the SEM of three separate real-time PCR measurements.

    Techniques Used: Methylation, CRISPR, Chromatin Immunoprecipitation, Infection, Real-time Polymerase Chain Reaction

    PRC2 is required for silent integration of HIV-1 in Jurkat T cells. (A) Experimental scheme. (B) Representative flow data measuring levels of H3K27me3 and HIV infection in cells expressing EZH2 or scrambled shRNA in untreated cells. (C) Cells treated with 1 µM SAHA. Black, cell counts per quadrant; red, distribution of GFP + cells in high-H3K27me3 gate; blue, distribution of GFP + cells in low-H3K27me3 gate. (D) Relative silencing of HIV-1 from cells treated with shRNAs targeting different subunits of PRC2. (Top) Inducible virus in high- and low-H3K27me3 populations. (Bottom) Relative silencing after knockdown of each of the PRC2 subunits. Error bars represent the SEM of three separate experiments.
    Figure Legend Snippet: PRC2 is required for silent integration of HIV-1 in Jurkat T cells. (A) Experimental scheme. (B) Representative flow data measuring levels of H3K27me3 and HIV infection in cells expressing EZH2 or scrambled shRNA in untreated cells. (C) Cells treated with 1 µM SAHA. Black, cell counts per quadrant; red, distribution of GFP + cells in high-H3K27me3 gate; blue, distribution of GFP + cells in low-H3K27me3 gate. (D) Relative silencing of HIV-1 from cells treated with shRNAs targeting different subunits of PRC2. (Top) Inducible virus in high- and low-H3K27me3 populations. (Bottom) Relative silencing after knockdown of each of the PRC2 subunits. Error bars represent the SEM of three separate experiments.

    Techniques Used: Flow Cytometry, Infection, Expressing, shRNA

    EZH2 and EHMT2 are required for the establishment of latent HIV-1 in primary Th17 cells. (A) Experimental design. (B) Reactivation of latent proviruses by Dynabeads Human T-Activator CD3/CD28, IL-15 (50 ng/ml), or ConA (5 µg/ml) in cells pretreated with DMSO, 100 nM GSK-343, EPZ-6438, or UNC-0638.
    Figure Legend Snippet: EZH2 and EHMT2 are required for the establishment of latent HIV-1 in primary Th17 cells. (A) Experimental design. (B) Reactivation of latent proviruses by Dynabeads Human T-Activator CD3/CD28, IL-15 (50 ng/ml), or ConA (5 µg/ml) in cells pretreated with DMSO, 100 nM GSK-343, EPZ-6438, or UNC-0638.

    Techniques Used:

    CRISPR-mediated depletion of EZH2 or JARID2 reactivates latent HIV-1 in E4 cells. Panels: A, EZH2-disrupted cells; B, JARID2-disrupted cells; C, EHMT2 (G9a)-disrupted cells. (Top) Western blot assays showing histone methyltransferases and methylated histones. (Bottom) Quantification of HIV-1 reactivation. E4 cells were infected with CRISPR-Cas9-expressing viruses targeting the proteins indicated. Reactivation of HIV-1 by stimulation with SAHA (1 μM) overnight was measured 7 days postinfection by FACS analysis. Error bars represent the SEM of three separate experiments.
    Figure Legend Snippet: CRISPR-mediated depletion of EZH2 or JARID2 reactivates latent HIV-1 in E4 cells. Panels: A, EZH2-disrupted cells; B, JARID2-disrupted cells; C, EHMT2 (G9a)-disrupted cells. (Top) Western blot assays showing histone methyltransferases and methylated histones. (Bottom) Quantification of HIV-1 reactivation. E4 cells were infected with CRISPR-Cas9-expressing viruses targeting the proteins indicated. Reactivation of HIV-1 by stimulation with SAHA (1 μM) overnight was measured 7 days postinfection by FACS analysis. Error bars represent the SEM of three separate experiments.

    Techniques Used: CRISPR, Western Blot, Methylation, Infection, Expressing, FACS

    40) Product Images from "Long noncoding RNA LINC01296 plays an oncogenic role in colorectal cancer by suppressing p15 expression"

    Article Title: Long noncoding RNA LINC01296 plays an oncogenic role in colorectal cancer by suppressing p15 expression

    Journal: The Journal of International Medical Research

    doi: 10.1177/03000605211004414

    LINC01296 represses p15 gene expression by interacting with EZH2. (a) Fluorescence in situ hybridization analysis of subcellular distribution of LINC01296 in SW480 and LoVo cells. Red, LINC01296 probe signal; blue, DAPI signal. (b) Cytoplasmic:nuclear expression ratios of LINC01296 in SW480 and LoVo cells detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (c) RNA immunoprecipitation results showed relative enrichment of LINC01296 RNA bound to EZH2 compared with levels bound to IgG. (d) Relative EZH2 and p15 mRNA expression levels in SW480 and LoVo cells transfected with EZH2 small interfering RNA (siRNA) detected by qRT-PCR. (e) Relative EZH2 and p15 protein expression levels in SW480 and LoVo cells transfected with EZH2 siRNA or LINC01296 antisense oligonucleotides detected by western blotting. (f) Chromatin immunoprecipitation assay demonstrated that LINC01296 knockdown decreased EZH2 binding and H3K27me3 in the p15 promoter. ** P
    Figure Legend Snippet: LINC01296 represses p15 gene expression by interacting with EZH2. (a) Fluorescence in situ hybridization analysis of subcellular distribution of LINC01296 in SW480 and LoVo cells. Red, LINC01296 probe signal; blue, DAPI signal. (b) Cytoplasmic:nuclear expression ratios of LINC01296 in SW480 and LoVo cells detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (c) RNA immunoprecipitation results showed relative enrichment of LINC01296 RNA bound to EZH2 compared with levels bound to IgG. (d) Relative EZH2 and p15 mRNA expression levels in SW480 and LoVo cells transfected with EZH2 small interfering RNA (siRNA) detected by qRT-PCR. (e) Relative EZH2 and p15 protein expression levels in SW480 and LoVo cells transfected with EZH2 siRNA or LINC01296 antisense oligonucleotides detected by western blotting. (f) Chromatin immunoprecipitation assay demonstrated that LINC01296 knockdown decreased EZH2 binding and H3K27me3 in the p15 promoter. ** P

    Techniques Used: Expressing, Fluorescence, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation, Transfection, Small Interfering RNA, Western Blot, Chromatin Immunoprecipitation, Binding Assay

    Related Articles

    Sonication:

    Article Title: Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells
    Article Snippet: .. Next, the cell debris was sonicated to generate 200- to 300-bp chromatin pieces and then compounded with anti-EZH2, anti-LSD1, anti-H3K27, anti-H3K4 (as positive reference), and IgG (as negative control) antibody-magnetic beads for immunoprecipitation. ..

    Negative Control:

    Article Title: Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells
    Article Snippet: .. Next, the cell debris was sonicated to generate 200- to 300-bp chromatin pieces and then compounded with anti-EZH2, anti-LSD1, anti-H3K27, anti-H3K4 (as positive reference), and IgG (as negative control) antibody-magnetic beads for immunoprecipitation. ..

    Article Title: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma
    Article Snippet: .. Briefly, cell extracts were prepared in RIP buffer and incubated with magnetic beads conjugated with control IgG (negative control), anti‐EZH2, or anti‐LSD1. ..

    Immunoprecipitation:

    Article Title: Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells
    Article Snippet: .. Next, the cell debris was sonicated to generate 200- to 300-bp chromatin pieces and then compounded with anti-EZH2, anti-LSD1, anti-H3K27, anti-H3K4 (as positive reference), and IgG (as negative control) antibody-magnetic beads for immunoprecipitation. ..

    Article Title: Long noncoding RNA UPK1A-AS1 indicates poor prognosis of hepatocellular carcinoma and promotes cell proliferation through interaction with EZH2
    Article Snippet: .. Cells were lysed with lysis buffer, and cell lysates were immunoprecipitated with anti-EZH2 and immunoglobulin (Ig) G antibodies. ..

    Article Title: A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma
    Article Snippet: .. For each immunoprecipitation reaction, the chromatin of 200,000 cells was combined with either 5 µg anti-H3K27me3 (Active Motif; cat. no 39155), 5 µg anti-IgG Rabbit (Millipore, cat. no PP64B), 1 µg anti-EZH2 (Millipore, cat. no 17-662) or 1 µg anti-IgG Mouse (Millipore; CS200621) antibodies. ..

    Article Title: Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis
    Article Snippet: .. Chromatin was immunoprecipitated at 4°C overnight with anti-JMJD3, anti-EZH2, anti-H3K27me3, or normal IgG as a NC. ..

    Lysis:

    Article Title: Long noncoding RNA UPK1A-AS1 indicates poor prognosis of hepatocellular carcinoma and promotes cell proliferation through interaction with EZH2
    Article Snippet: .. Cells were lysed with lysis buffer, and cell lysates were immunoprecipitated with anti-EZH2 and immunoglobulin (Ig) G antibodies. ..

    Transfection:

    Article Title: Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci
    Article Snippet: .. The eluted fraction was loaded on 4-15% gradient gels with Input and immunoblotted with anti-HA, anti-KAT5, anti-DMAP1, anti-SUZ12 or anti-EZH2 antibody as appropriate., : EPC1-PHF1-Flag tagged K562 Cells and controls were grown in 100mm dishes, transiently transfected with JAZF1-full length or JAZF1 (1-281) Myc tagged constructs. ..

    Construct:

    Article Title: Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci
    Article Snippet: .. The eluted fraction was loaded on 4-15% gradient gels with Input and immunoblotted with anti-HA, anti-KAT5, anti-DMAP1, anti-SUZ12 or anti-EZH2 antibody as appropriate., : EPC1-PHF1-Flag tagged K562 Cells and controls were grown in 100mm dishes, transiently transfected with JAZF1-full length or JAZF1 (1-281) Myc tagged constructs. ..

    Incubation:

    Article Title: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma
    Article Snippet: .. Briefly, cell extracts were prepared in RIP buffer and incubated with magnetic beads conjugated with control IgG (negative control), anti‐EZH2, or anti‐LSD1. ..

    Magnetic Beads:

    Article Title: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma
    Article Snippet: .. Briefly, cell extracts were prepared in RIP buffer and incubated with magnetic beads conjugated with control IgG (negative control), anti‐EZH2, or anti‐LSD1. ..

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  • 99
    Millipore antibodies against ezh2
    PVT1 Binds with <t>EZH2</t> in the Nucleus and Epigenetically Silences ANGPTL4 , Inhibiting Cell Proliferation and Migration in CCA Cell Lines (A) Expression level of ANGPTL4 in cholangiocarcinoma based on the analysis of GEO: GSE26566 data. (B) ANGPTL4 expression was determined in 17 pairs of CCA tissues by qRT-PCR. (C–F) HuCCT1 and RBE cells transfected with vector/ ANGPTL4 /pcDNA- PVT1 and cells transfected with PVT1 followed by transfection with ANGPTL4 . After transfection, the cells were analyzed by CCK-8 assays (C and D) and transwell assays (E and F). (G and H) ChIP of EZH2 and H3K27me3 of the promoter region of the ANGPTL4 locus after siRNA treatment with si-NC and si- PVT1 2# (G) or overexpression of PVT1 (H) in HuCCT1 cells. qPCR was performed to quantify the ChIP assay products. Enrichment was quantified relative to the input controls. Antibodies directed against IgG were used as a negative control. (I) Proposed model by which PVT1 regulates ANGPTL4 expression to promote CCA tumor growth. The error bars indicate the means ± SD. *p
    Antibodies Against Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore anti ezh2 antibodies
    LncLALC recruited DNA methyltransferases to the LZTS1 promoter regions by combining with <t>EZH2.</t> A Preliminary prediction by bioinformatics system showed that Lnc-LALC may have certain binding ability to EZH2. ( http://service.tartaglialab.com/page/catrapid_group ). B , C RIP test using the EZH2 antibody showed that there was a strong combining capacity between lncLALC and EZH2 in SW480 cells and LOVO cells. D qRT-PCR showed that EZH2 was elevated in CRC cells than NCM460 cells. E The silencing efficacy of EZH2 in SW480 cells was determined by qRT-PCR. F The CHIP results showed that siEZH2 could reduce the enrichment of DNMT1, DNMT3A, DNMT3B in LZTS1 promoter and could also reverse the DNMTs , high enrichment induced by Lv-lncLALC in SW480 cells. G qRT-PCR showed that siEZH2 could increase LZTS1 expression level and abolish the suppression of LZTS1 induced by Lv-lncLALC in SW480 cells. * P
    Anti Ezh2 Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ezh2 antibodies/product/Millipore
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    Millipore ezh2 specific antibody
    ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of <t>EZH2</t> to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P
    Ezh2 Specific Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 specific antibody/product/Millipore
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    PVT1 Binds with EZH2 in the Nucleus and Epigenetically Silences ANGPTL4 , Inhibiting Cell Proliferation and Migration in CCA Cell Lines (A) Expression level of ANGPTL4 in cholangiocarcinoma based on the analysis of GEO: GSE26566 data. (B) ANGPTL4 expression was determined in 17 pairs of CCA tissues by qRT-PCR. (C–F) HuCCT1 and RBE cells transfected with vector/ ANGPTL4 /pcDNA- PVT1 and cells transfected with PVT1 followed by transfection with ANGPTL4 . After transfection, the cells were analyzed by CCK-8 assays (C and D) and transwell assays (E and F). (G and H) ChIP of EZH2 and H3K27me3 of the promoter region of the ANGPTL4 locus after siRNA treatment with si-NC and si- PVT1 2# (G) or overexpression of PVT1 (H) in HuCCT1 cells. qPCR was performed to quantify the ChIP assay products. Enrichment was quantified relative to the input controls. Antibodies directed against IgG were used as a negative control. (I) Proposed model by which PVT1 regulates ANGPTL4 expression to promote CCA tumor growth. The error bars indicate the means ± SD. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Long Non-coding RNA PVT1 Promotes Cell Proliferation and Migration by Silencing ANGPTL4 Expression in Cholangiocarcinoma

    doi: 10.1016/j.omtn.2018.10.001

    Figure Lengend Snippet: PVT1 Binds with EZH2 in the Nucleus and Epigenetically Silences ANGPTL4 , Inhibiting Cell Proliferation and Migration in CCA Cell Lines (A) Expression level of ANGPTL4 in cholangiocarcinoma based on the analysis of GEO: GSE26566 data. (B) ANGPTL4 expression was determined in 17 pairs of CCA tissues by qRT-PCR. (C–F) HuCCT1 and RBE cells transfected with vector/ ANGPTL4 /pcDNA- PVT1 and cells transfected with PVT1 followed by transfection with ANGPTL4 . After transfection, the cells were analyzed by CCK-8 assays (C and D) and transwell assays (E and F). (G and H) ChIP of EZH2 and H3K27me3 of the promoter region of the ANGPTL4 locus after siRNA treatment with si-NC and si- PVT1 2# (G) or overexpression of PVT1 (H) in HuCCT1 cells. qPCR was performed to quantify the ChIP assay products. Enrichment was quantified relative to the input controls. Antibodies directed against IgG were used as a negative control. (I) Proposed model by which PVT1 regulates ANGPTL4 expression to promote CCA tumor growth. The error bars indicate the means ± SD. *p

    Article Snippet: Antibodies against EZH2 were purchased from Millipore (Billerica, MA, USA), and antibodies against H3 trimethyl Lys27 (H3K27me3) were purchased from Abcam (Cambridge, UK).

    Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, CCK-8 Assay, Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction, Negative Control

    PVT1 Binds with EZH2 to Coregulate Target Genes, Especially ANGPTL4 (A) After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosolic marker and U1 was used as a nuclear marker. (B) Fluorescent images of RBE cells treated with anti- PVT1 (red), anti- 18S (red), and anti- U6 (red) RNA probes. DAPI staining is shown in blue. (C) The probability of interaction of EZH2 and PVT1 was determined with an online tool ( http://pridb.gdcb.iastate.edu/RPISeq/index.html ). Predictions with probabilities > 0.5 were considered positive. RPI-seq predictions are based on random forest (RF) or support vector machine (SVM). (D) An RIP experiment for EZH2 was performed, and the coprecipitated RNA was subjected to qRT-PCR for PVT1 . (E) Expression and co-localization of EZH2 and PVT1 in HuCCT1 cells. Representative fluorescent images show HuCCT1 cells treated with fluorescently labeled anti-EZH2 antibody (green) and anti- PVT1 RNA (red). DAPI staining indicates the cell nuclei (blue). (F) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after knockdown of EZH2 . (G) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after overexpression of EZH2 . (H) The correlation between EZH2 and ANGPTL4 expression was detected by analyzing GEO: GSE26566 data. (I) The altered protein levels of ANGPTL4 were selectively confirmed by western blot analysis in cells with knockdown of PVT1 or EZH2 . (J) The altered protein levels of ANGPTL4 were selectively confirmed by western blotting in cells overexpressing PVT1 or EZH2 . The error bars indicate the means ± SD. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Long Non-coding RNA PVT1 Promotes Cell Proliferation and Migration by Silencing ANGPTL4 Expression in Cholangiocarcinoma

    doi: 10.1016/j.omtn.2018.10.001

    Figure Lengend Snippet: PVT1 Binds with EZH2 to Coregulate Target Genes, Especially ANGPTL4 (A) After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosolic marker and U1 was used as a nuclear marker. (B) Fluorescent images of RBE cells treated with anti- PVT1 (red), anti- 18S (red), and anti- U6 (red) RNA probes. DAPI staining is shown in blue. (C) The probability of interaction of EZH2 and PVT1 was determined with an online tool ( http://pridb.gdcb.iastate.edu/RPISeq/index.html ). Predictions with probabilities > 0.5 were considered positive. RPI-seq predictions are based on random forest (RF) or support vector machine (SVM). (D) An RIP experiment for EZH2 was performed, and the coprecipitated RNA was subjected to qRT-PCR for PVT1 . (E) Expression and co-localization of EZH2 and PVT1 in HuCCT1 cells. Representative fluorescent images show HuCCT1 cells treated with fluorescently labeled anti-EZH2 antibody (green) and anti- PVT1 RNA (red). DAPI staining indicates the cell nuclei (blue). (F) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after knockdown of EZH2 . (G) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after overexpression of EZH2 . (H) The correlation between EZH2 and ANGPTL4 expression was detected by analyzing GEO: GSE26566 data. (I) The altered protein levels of ANGPTL4 were selectively confirmed by western blot analysis in cells with knockdown of PVT1 or EZH2 . (J) The altered protein levels of ANGPTL4 were selectively confirmed by western blotting in cells overexpressing PVT1 or EZH2 . The error bars indicate the means ± SD. *p

    Article Snippet: Antibodies against EZH2 were purchased from Millipore (Billerica, MA, USA), and antibodies against H3 trimethyl Lys27 (H3K27me3) were purchased from Abcam (Cambridge, UK).

    Techniques: RNA Expression, Quantitative RT-PCR, Marker, Staining, Plasmid Preparation, Expressing, Labeling, Methylation, Over Expression, Western Blot

    Ezh2 and the circadian clock regulate zebrafish hematopoietic genes. ( A–D ) qRT-PCR analysis of cmyb (A, B) and lck (C, D) in WT and ezh2–/– mutant under DD (A, C) and LD (B, D) conditions. Three independent experiments were conducted. Rhythmic mRNA expression was analyzed with the JTK-CYCLE method. ADJ.P for adjusted minimal P -values ( P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Ezh2 and the circadian clock regulate zebrafish hematopoietic genes. ( A–D ) qRT-PCR analysis of cmyb (A, B) and lck (C, D) in WT and ezh2–/– mutant under DD (A, C) and LD (B, D) conditions. Three independent experiments were conducted. Rhythmic mRNA expression was analyzed with the JTK-CYCLE method. ADJ.P for adjusted minimal P -values ( P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Quantitative RT-PCR, Mutagenesis, Expressing

    Zebrafish ezh2 is a circadian clock-controlled gene. ( A, B ) ezh2 is expressed rhythmically in developing zebrafish embryos under DD (A) and LD (B) conditions. ( C–F ) Up-regulation of ezh2 in per1b –/– (C, D) and rev-erbα –/– (E, F) mutant zebrafish under DD (C, E) and LD (D, F) conditions. For (A) to (F), at least three independent experiments were performed. Levels of mRNA expression were analyzed with the JTK-CYCLE method. ADJ.P for adjusted minimal P -values ( P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Zebrafish ezh2 is a circadian clock-controlled gene. ( A, B ) ezh2 is expressed rhythmically in developing zebrafish embryos under DD (A) and LD (B) conditions. ( C–F ) Up-regulation of ezh2 in per1b –/– (C, D) and rev-erbα –/– (E, F) mutant zebrafish under DD (C, E) and LD (D, F) conditions. For (A) to (F), at least three independent experiments were performed. Levels of mRNA expression were analyzed with the JTK-CYCLE method. ADJ.P for adjusted minimal P -values ( P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Mutagenesis, Expressing

    Altered locomotor rhythms in ezh2 morphants and ezh2 -overexpressing zebrafish larvae. ( A–H ) Locomotor activities were monitored and analyzed in ezh2 morphants and control zebrafish larvae under DD (A–D) or LD (E–H) condition. The period (B), amplitude (C) and phase (D) under DD condition and total average moving distances (F), at day (G) and night (H) under LD conditions were shown. Student's t -test was conducted. * P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Altered locomotor rhythms in ezh2 morphants and ezh2 -overexpressing zebrafish larvae. ( A–H ) Locomotor activities were monitored and analyzed in ezh2 morphants and control zebrafish larvae under DD (A–D) or LD (E–H) condition. The period (B), amplitude (C) and phase (D) under DD condition and total average moving distances (F), at day (G) and night (H) under LD conditions were shown. Student's t -test was conducted. * P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques:

    Zebrafish Ezh2 enhances expression of clock genes through binding to core clock components independent of its H3K27 methyltrasnsferase activity. ( A, B ) Co-IP assays show that zebrafish Ezh2 complexes with Bmal1b, Clock1a, and Cry1aa. ( C ) Luciferase reporter assays show that zebrafish Ezh2 enhances transcription activities of Clock1a and Bmal1b in a dose-dependent manner. Co-transfection was performed as indicated, and luciferase activities were assayed 24 h after transfection. Experiments were performed in three independent experiments. Transactivation activities are expressed as fold increase over the control group. Values are expressed as means ± S.D., n = 3. Student's t -test was conducted. * P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Zebrafish Ezh2 enhances expression of clock genes through binding to core clock components independent of its H3K27 methyltrasnsferase activity. ( A, B ) Co-IP assays show that zebrafish Ezh2 complexes with Bmal1b, Clock1a, and Cry1aa. ( C ) Luciferase reporter assays show that zebrafish Ezh2 enhances transcription activities of Clock1a and Bmal1b in a dose-dependent manner. Co-transfection was performed as indicated, and luciferase activities were assayed 24 h after transfection. Experiments were performed in three independent experiments. Transactivation activities are expressed as fold increase over the control group. Values are expressed as means ± S.D., n = 3. Student's t -test was conducted. * P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Expressing, Binding Assay, Activity Assay, Co-Immunoprecipitation Assay, Luciferase, Cotransfection, Transfection

    Generation and characterization of a zebrafish ezh2 null mutant. ( A ) The C to T point mutation in the ezh2 coding region was confirmed by DNA sequencing. WT, wild-type. ( B ) Gene structure of ezh2 and the point mutation occurs in Exon 2. ( C ) The C to T nonsense mutation results in a truncated peptide and losses of its major functional domains. SANT, SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains; CXC, Tesmin/TSO1-like CXC domain; SET, Su(var)3–9, Enhancer-of-zeste, Trithorax domain. ( D ) Images of WT and ezh2–/– mutant zebrafish from 24 hpf to 72 hpf. Note that the ezh2–/– mutant zebrafish display curved tail and thin yolk sac extension around 28 hpf and short body length and small eyes after 48 hpf. Scale bar , 0.5 mm. ( E ) Western Blotting shows that expression of Ezh2 is abolished and that of H3K27 mono-, di- and trimethylation are reduced in ezh2–/– mutant fish. Proteins of zebrafish larvae were extracted with RIPA buffer at 48 hpf. Western Bloting was performed with indicated antibodies. ( F ) Quantification of western bloting images shown in (E) with ImageJ. Student's t -test was conducted. *** P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Generation and characterization of a zebrafish ezh2 null mutant. ( A ) The C to T point mutation in the ezh2 coding region was confirmed by DNA sequencing. WT, wild-type. ( B ) Gene structure of ezh2 and the point mutation occurs in Exon 2. ( C ) The C to T nonsense mutation results in a truncated peptide and losses of its major functional domains. SANT, SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains; CXC, Tesmin/TSO1-like CXC domain; SET, Su(var)3–9, Enhancer-of-zeste, Trithorax domain. ( D ) Images of WT and ezh2–/– mutant zebrafish from 24 hpf to 72 hpf. Note that the ezh2–/– mutant zebrafish display curved tail and thin yolk sac extension around 28 hpf and short body length and small eyes after 48 hpf. Scale bar , 0.5 mm. ( E ) Western Blotting shows that expression of Ezh2 is abolished and that of H3K27 mono-, di- and trimethylation are reduced in ezh2–/– mutant fish. Proteins of zebrafish larvae were extracted with RIPA buffer at 48 hpf. Western Bloting was performed with indicated antibodies. ( F ) Quantification of western bloting images shown in (E) with ImageJ. Student's t -test was conducted. *** P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Mutagenesis, DNA Sequencing, Functional Assay, Binding Assay, Western Blot, Expressing, Fluorescence In Situ Hybridization

    Primitive hematopoiesis is disrupted in ezh2–/– mutant zebrafish. (A, B ) O-dianisidine (O-d) staining of wild-type sibling (A) and ezh2 –/– mutant fish (B) at 33 hpf. Black arrowhead indicates hemoglobin-staining, which is significantly reduced in ezh2–/– mutant zebrafish. ( C–L ) In situ hybridization of wild-type (C, E, G, I and K) and ezh2–/– mutant embryos (D, F, H, J and L) using probes of gata1 ( C, D ), pu.1 ( E, F ), hbbe1 ( G, H ), mpx ( I, J ), l-plastin ( K, L ) at 26 hpf ( C-F ) and 28 hpf ( G-L ), respectively. White arrowheads in ( C ) and ( G ) indicate ICM (Interme diate cell mass) and black arrowheads in (E, I and K) PBI (Posterior blood island). Scale bar , 0.25 mm. ( M–P ) Images of gata1:dsRed cells in WT (M, O) and ezh2–/– mutant (N, P) fish at 24 hpf (M, N) and 28 hpf (O, P). ( Q–T ) Images of mpx:eGFP cells in wild-type (Q, S) and ezh2–/– mutant (R, T) fish at 28 hpf (Q, R) and 48 hpf (S, T). ( U, V ) Images of lyz:eGFP cells in wild-type (U) and ezh2–/– mutant (V) fish at 28 hpf. White arrowheads in (M–R) indicate ICM and red arrowheads in (S–V) PBI. Scale bar in (M–V), 0.25 mm. All images shown are lateral view, anterior to left.

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Primitive hematopoiesis is disrupted in ezh2–/– mutant zebrafish. (A, B ) O-dianisidine (O-d) staining of wild-type sibling (A) and ezh2 –/– mutant fish (B) at 33 hpf. Black arrowhead indicates hemoglobin-staining, which is significantly reduced in ezh2–/– mutant zebrafish. ( C–L ) In situ hybridization of wild-type (C, E, G, I and K) and ezh2–/– mutant embryos (D, F, H, J and L) using probes of gata1 ( C, D ), pu.1 ( E, F ), hbbe1 ( G, H ), mpx ( I, J ), l-plastin ( K, L ) at 26 hpf ( C-F ) and 28 hpf ( G-L ), respectively. White arrowheads in ( C ) and ( G ) indicate ICM (Interme diate cell mass) and black arrowheads in (E, I and K) PBI (Posterior blood island). Scale bar , 0.25 mm. ( M–P ) Images of gata1:dsRed cells in WT (M, O) and ezh2–/– mutant (N, P) fish at 24 hpf (M, N) and 28 hpf (O, P). ( Q–T ) Images of mpx:eGFP cells in wild-type (Q, S) and ezh2–/– mutant (R, T) fish at 28 hpf (Q, R) and 48 hpf (S, T). ( U, V ) Images of lyz:eGFP cells in wild-type (U) and ezh2–/– mutant (V) fish at 28 hpf. White arrowheads in (M–R) indicate ICM and red arrowheads in (S–V) PBI. Scale bar in (M–V), 0.25 mm. All images shown are lateral view, anterior to left.

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Mutagenesis, Staining, Fluorescence In Situ Hybridization, In Situ Hybridization

    Definitive hematopoiesis is disrupted in ezh2–/– mutant zebrafish. ( A–L ) In situ hybridization of WT sibling (A, C, E, G, I and K) and ezh2–/– mutant embryos or larvae (B, D, F, G, J and L) using probes of runx1 (A, B), cmyb (C, D), ikaros (E, H), lck (I, J), rag-1 (K, L) at 28 hpf (A, F) and 96 hpf (G, L), respectively. White arrowheads indicate ICM in A, C and E and black arrowheads the thymus in G, I and K. Scale bar , 0.25 mm. ( M–P ) Images of cmyb:eGFP cells in wild-type ( M, O ) and ezh2–/– mutant (N, P) fish at 32 hpf ( M, N ) and 72 hpf (O, P). ( Q–T ) Images of coro1a:eGFP cells in wild-type (Q, S) and ezh2–/– mutant (R, T) fish at 28 hpf (Q, R) and 72 hpf (S, T). White arrowheads indicate ICM in M, N, Q and R and dashed circle the thymus in O, P, S and T. Scale bar in (M–T), 0.25 mm. All images shown are lateral view, anterior to left.

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Definitive hematopoiesis is disrupted in ezh2–/– mutant zebrafish. ( A–L ) In situ hybridization of WT sibling (A, C, E, G, I and K) and ezh2–/– mutant embryos or larvae (B, D, F, G, J and L) using probes of runx1 (A, B), cmyb (C, D), ikaros (E, H), lck (I, J), rag-1 (K, L) at 28 hpf (A, F) and 96 hpf (G, L), respectively. White arrowheads indicate ICM in A, C and E and black arrowheads the thymus in G, I and K. Scale bar , 0.25 mm. ( M–P ) Images of cmyb:eGFP cells in wild-type ( M, O ) and ezh2–/– mutant (N, P) fish at 32 hpf ( M, N ) and 72 hpf (O, P). ( Q–T ) Images of coro1a:eGFP cells in wild-type (Q, S) and ezh2–/– mutant (R, T) fish at 28 hpf (Q, R) and 72 hpf (S, T). White arrowheads indicate ICM in M, N, Q and R and dashed circle the thymus in O, P, S and T. Scale bar in (M–T), 0.25 mm. All images shown are lateral view, anterior to left.

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Mutagenesis, In Situ Hybridization, Fluorescence In Situ Hybridization

    Disrupted expression of hematopoietic genes in ezh2–/– mutant zebrafish revealed by transcriptome analysis. ( A ) Numbers of differentially expressed genes (DEGs) in ezh2–/– mutant zebrafish at 28 hpf, revealed by transcriptome analysis. ( B ) Histogram of 10 hematopoietic DEGs in ezh2–/– mutant zebrafish. Red and green colors represent up-regulation and down-regulation, respectively. ( C ) Nine out of 10 down-regulated hematopoietic genes revealed by transcriptome analysis are reconfirmed by independent qRT-PCR analysis. Three independent experiments were performed. Statistical analysis was performed using Student's t -test. * P

    Journal: Nucleic Acids Research

    Article Title: Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

    doi: 10.1093/nar/gky101

    Figure Lengend Snippet: Disrupted expression of hematopoietic genes in ezh2–/– mutant zebrafish revealed by transcriptome analysis. ( A ) Numbers of differentially expressed genes (DEGs) in ezh2–/– mutant zebrafish at 28 hpf, revealed by transcriptome analysis. ( B ) Histogram of 10 hematopoietic DEGs in ezh2–/– mutant zebrafish. Red and green colors represent up-regulation and down-regulation, respectively. ( C ) Nine out of 10 down-regulated hematopoietic genes revealed by transcriptome analysis are reconfirmed by independent qRT-PCR analysis. Three independent experiments were performed. Statistical analysis was performed using Student's t -test. * P

    Article Snippet: Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).

    Techniques: Expressing, Mutagenesis, Quantitative RT-PCR

    LncLALC recruited DNA methyltransferases to the LZTS1 promoter regions by combining with EZH2. A Preliminary prediction by bioinformatics system showed that Lnc-LALC may have certain binding ability to EZH2. ( http://service.tartaglialab.com/page/catrapid_group ). B , C RIP test using the EZH2 antibody showed that there was a strong combining capacity between lncLALC and EZH2 in SW480 cells and LOVO cells. D qRT-PCR showed that EZH2 was elevated in CRC cells than NCM460 cells. E The silencing efficacy of EZH2 in SW480 cells was determined by qRT-PCR. F The CHIP results showed that siEZH2 could reduce the enrichment of DNMT1, DNMT3A, DNMT3B in LZTS1 promoter and could also reverse the DNMTs , high enrichment induced by Lv-lncLALC in SW480 cells. G qRT-PCR showed that siEZH2 could increase LZTS1 expression level and abolish the suppression of LZTS1 induced by Lv-lncLALC in SW480 cells. * P

    Journal: Cell Death & Disease

    Article Title: Long non-coding RNA Lnc-LALC facilitates colorectal cancer liver metastasis via epigenetically silencing LZTS1

    doi: 10.1038/s41419-021-03461-w

    Figure Lengend Snippet: LncLALC recruited DNA methyltransferases to the LZTS1 promoter regions by combining with EZH2. A Preliminary prediction by bioinformatics system showed that Lnc-LALC may have certain binding ability to EZH2. ( http://service.tartaglialab.com/page/catrapid_group ). B , C RIP test using the EZH2 antibody showed that there was a strong combining capacity between lncLALC and EZH2 in SW480 cells and LOVO cells. D qRT-PCR showed that EZH2 was elevated in CRC cells than NCM460 cells. E The silencing efficacy of EZH2 in SW480 cells was determined by qRT-PCR. F The CHIP results showed that siEZH2 could reduce the enrichment of DNMT1, DNMT3A, DNMT3B in LZTS1 promoter and could also reverse the DNMTs , high enrichment induced by Lv-lncLALC in SW480 cells. G qRT-PCR showed that siEZH2 could increase LZTS1 expression level and abolish the suppression of LZTS1 induced by Lv-lncLALC in SW480 cells. * P

    Article Snippet: CRC cells were collected and lysed by the RIP lysis buffer, then incubated with the magnetic beds and anti-EZH2 antibodies overnight at 4 °C.

    Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Expressing

    ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P

    Journal: Cancer Management and Research

    Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion

    doi: 10.2147/CMAR.S186777

    Figure Lengend Snippet: ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P

    Article Snippet: ChIP assay was carried out using the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (EMD Millipore) and EZH2-specific antibody (EMD Millipore) or H3K27me3-specific antibody (EMD Millipore) according to the instructions.

    Techniques: Expressing, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Western Blot