ezh2  (Millipore)


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  • 97
    Name:
    O Meara s Reagent
    Description:

    Catalog Number:
    07689
    Price:
    None
    Applications:
    The reagent is used in Voges-Proskauer test for the detection of acetoin production by bacterial cultures.
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    Structured Review

    Millipore ezh2
    LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to <t>EZH2</t> and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p

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    ezh2 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Long Noncoding RNA LINC00673 Is Activated by SP1 and Exerts Oncogenic Properties by Interacting with LSD1 and EZH2 in Gastric Cancer"

    Article Title: Long Noncoding RNA LINC00673 Is Activated by SP1 and Exerts Oncogenic Properties by Interacting with LSD1 and EZH2 in Gastric Cancer

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.01.017

    LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to EZH2 and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p
    Figure Legend Snippet: LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to EZH2 and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p

    Techniques Used: Binding Assay, Fluorescence In Situ Hybridization, Quantitative RT-PCR, RNA Expression, Western Blot, Positive Control, Expressing, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    2) Product Images from "EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer"

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr2214

    EZH2 and BMI1 inversely correlate with variables associated with survival . (a) EZH2 and BMI1 distribution differ by grade. Mean EZH2 mRNA levels are -0.62 (n = 75), -0.21 (n = 101) and 0.37 (n = 119) for grades 1, 2 and 3, respectively (p
    Figure Legend Snippet: EZH2 and BMI1 inversely correlate with variables associated with survival . (a) EZH2 and BMI1 distribution differ by grade. Mean EZH2 mRNA levels are -0.62 (n = 75), -0.21 (n = 101) and 0.37 (n = 119) for grades 1, 2 and 3, respectively (p

    Techniques Used:

    Model for the different oncogenic roles of EZH2 and BMI1 . (a) In normal development, genes such as the INK4a/ARF locus are bound by EZH2, marked by H3K27me3 and bound by BMI1. (b) On oncogene activation (indicated by lightning symbol) such as constitutive Ras or Myc expression, the INK4a/ARF locus becomes expressed as part of a tumour suppressive response. (c) Increased BMI1 expression results in more BMI1 protein binding to known Polycomb group (PcG)-target genes. This prevents activation of these genes even when signals are present that would normally activate these genes. (d) Increased EZH2 expression does not prevent INK4a/ARF activation (although this locus may still be inactivated by other mechanisms). In contrast, the role of EZH2 in tumourigenesis may be due to silencing of genes not normally targeted by PcG or due to methylation of non-histone proteins (indicated by diamond on protein 'X').
    Figure Legend Snippet: Model for the different oncogenic roles of EZH2 and BMI1 . (a) In normal development, genes such as the INK4a/ARF locus are bound by EZH2, marked by H3K27me3 and bound by BMI1. (b) On oncogene activation (indicated by lightning symbol) such as constitutive Ras or Myc expression, the INK4a/ARF locus becomes expressed as part of a tumour suppressive response. (c) Increased BMI1 expression results in more BMI1 protein binding to known Polycomb group (PcG)-target genes. This prevents activation of these genes even when signals are present that would normally activate these genes. (d) Increased EZH2 expression does not prevent INK4a/ARF activation (although this locus may still be inactivated by other mechanisms). In contrast, the role of EZH2 in tumourigenesis may be due to silencing of genes not normally targeted by PcG or due to methylation of non-histone proteins (indicated by diamond on protein 'X').

    Techniques Used: Activation Assay, Expressing, Protein Binding, Methylation

    EZH2 and BMI1 are inversely correlated . (a). Univariate survival analyses by Kaplan-Meier plots and log rank tests. Patients were categorised by high or low expression of the different Polycomb group (PcG) members based on microarray data. (b) Relative risk of death evaluated by Cox regression modelling and continous PcG mRNA levels. Multivariate analysis is adjusted for tumour size, lymph node (LN) status and grade. (c) Relative risk of death based on continous EZH2 and BMI1 levels in LN-positive and LN-negative patients. P het = p value indicating heterogeneity of BMI1 levels across LN-positive and LN-negative patients. (d) Two-by-two table for categorical EZH2 and BMI1 levels. Odds ratio (OR) = 0.55 (95% confidence interval (CI) = 0.33 to 0.90) for high EZH2 among patients with high BMI1 compared with low BMI1 (chi-squared: p = 0.017).
    Figure Legend Snippet: EZH2 and BMI1 are inversely correlated . (a). Univariate survival analyses by Kaplan-Meier plots and log rank tests. Patients were categorised by high or low expression of the different Polycomb group (PcG) members based on microarray data. (b) Relative risk of death evaluated by Cox regression modelling and continous PcG mRNA levels. Multivariate analysis is adjusted for tumour size, lymph node (LN) status and grade. (c) Relative risk of death based on continous EZH2 and BMI1 levels in LN-positive and LN-negative patients. P het = p value indicating heterogeneity of BMI1 levels across LN-positive and LN-negative patients. (d) Two-by-two table for categorical EZH2 and BMI1 levels. Odds ratio (OR) = 0.55 (95% confidence interval (CI) = 0.33 to 0.90) for high EZH2 among patients with high BMI1 compared with low BMI1 (chi-squared: p = 0.017).

    Techniques Used: Expressing, Microarray

    Correlation of EZH2 and BMI1 expression with molecular subtypes . (a) Mean mRNA levels of EZH2 and BMI1 in the different subgroups. The distribution of both EZH2 and BMI1 over the subgroups differ significantly, Kruskall-Wallis test. (b) Distribution of subgroups over categorised EZH2 and BMI1 levels. (c) Immunohistochemistry on EZH2 and BMI1 in normal breast tissue, an ErbB2-type and a luminal A-type tumour. Pictures were taken at 40× magnification (left panels) and a zoomed-in section is shown on the right of each image.
    Figure Legend Snippet: Correlation of EZH2 and BMI1 expression with molecular subtypes . (a) Mean mRNA levels of EZH2 and BMI1 in the different subgroups. The distribution of both EZH2 and BMI1 over the subgroups differ significantly, Kruskall-Wallis test. (b) Distribution of subgroups over categorised EZH2 and BMI1 levels. (c) Immunohistochemistry on EZH2 and BMI1 in normal breast tissue, an ErbB2-type and a luminal A-type tumour. Pictures were taken at 40× magnification (left panels) and a zoomed-in section is shown on the right of each image.

    Techniques Used: Expressing, Immunohistochemistry

    Differential effect of BMI1 and EZH2 on INK4a/ARF locus and association with TP53 mutations . (a) Dotplot of INK4a/ARF (CDKN2A) expression versus BMI1 and EZH2 expression. INK4a/ARF expression is not found in tumours with high BMI1 mRNA, whereas it does occur in tumours with high EZH2 (red circle). (b) Distribution of patients with positive TP53 staining (mutated TP53) differs by categorised EZH2 and BMI1 levels. TP53 immunohistochemistry (IHC) data was obtained from 273 patients and the TP53 sequence was analysed in 204 tumours. The distribution of mutant TP53 shows a similar pattern (TP53 mutation). (c) Logistic regression analysis of TP53 IHC and sequence status on EZH2 and BMI1 levels. CI = confidence interval, OR = odds ratio.
    Figure Legend Snippet: Differential effect of BMI1 and EZH2 on INK4a/ARF locus and association with TP53 mutations . (a) Dotplot of INK4a/ARF (CDKN2A) expression versus BMI1 and EZH2 expression. INK4a/ARF expression is not found in tumours with high BMI1 mRNA, whereas it does occur in tumours with high EZH2 (red circle). (b) Distribution of patients with positive TP53 staining (mutated TP53) differs by categorised EZH2 and BMI1 levels. TP53 immunohistochemistry (IHC) data was obtained from 273 patients and the TP53 sequence was analysed in 204 tumours. The distribution of mutant TP53 shows a similar pattern (TP53 mutation). (c) Logistic regression analysis of TP53 IHC and sequence status on EZH2 and BMI1 levels. CI = confidence interval, OR = odds ratio.

    Techniques Used: Expressing, Staining, Immunohistochemistry, Sequencing, Mutagenesis

    3) Product Images from "Long non-coding RNA LINC01133 represses KLF2, P21 and E-cadherin transcription through binding with EZH2, LSD1 in non small cell lung cancer"

    Article Title: Long non-coding RNA LINC01133 represses KLF2, P21 and E-cadherin transcription through binding with EZH2, LSD1 in non small cell lung cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7077

    LINC01133 interacted with EZH2 and LSD1, and silence KLF2, P21 and E-cadherin expression A. Relative LINC01133 levels in cell cytoplasm or Nucleus of NSCLC cell lines were detected by qPCR. B. RNA levels in immunoprecipitates were determined by qPCR. Expression levels of LINC01133 RNA were presented as fold enrichment relative to IgG immunoprecipitates. C. Protein levels in immunoprecipitates were determined by western blot. Expression levels of EZH2 and LSD1 protein were presented. D. The levels of KLF2, P21, PTEN, LATS2, RRAD, ASPP2 and E-cadherin mRNA were determined by qPCR when knockdown of LINC01133. E. The KLF2, P21 and E-cadherin protein levels were determined by western blot in LINC01133 knockdown A549 and PC9 cells. F. The E-cadherin protein levels were determined by immunofluorescence analysis in LINC01133 knockdown A549 cells. G. The KLF2, P21 and E-cadherin expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in A549 and PC9 cells. H, I. ChIP–qPCR of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding in the KLF2, P21 or E-cadherin promoter in A549 and PC9 cells, and IgG as a negative control. The mean values and s.d. were calculated from triplicates of a representative experiment.
    Figure Legend Snippet: LINC01133 interacted with EZH2 and LSD1, and silence KLF2, P21 and E-cadherin expression A. Relative LINC01133 levels in cell cytoplasm or Nucleus of NSCLC cell lines were detected by qPCR. B. RNA levels in immunoprecipitates were determined by qPCR. Expression levels of LINC01133 RNA were presented as fold enrichment relative to IgG immunoprecipitates. C. Protein levels in immunoprecipitates were determined by western blot. Expression levels of EZH2 and LSD1 protein were presented. D. The levels of KLF2, P21, PTEN, LATS2, RRAD, ASPP2 and E-cadherin mRNA were determined by qPCR when knockdown of LINC01133. E. The KLF2, P21 and E-cadherin protein levels were determined by western blot in LINC01133 knockdown A549 and PC9 cells. F. The E-cadherin protein levels were determined by immunofluorescence analysis in LINC01133 knockdown A549 cells. G. The KLF2, P21 and E-cadherin expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in A549 and PC9 cells. H, I. ChIP–qPCR of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding in the KLF2, P21 or E-cadherin promoter in A549 and PC9 cells, and IgG as a negative control. The mean values and s.d. were calculated from triplicates of a representative experiment.

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

    4) Product Images from "LncRNA TINCR attenuates cardiac hypertrophy by epigenetically silencing CaMKII"

    Article Title: LncRNA TINCR attenuates cardiac hypertrophy by epigenetically silencing CaMKII

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17735

    ( A ) The RIP assay was conducted to confirm whether TINCR could directly bind with EZH2. ( B ) The ChIP assay was performed to verify whether EZH2 could directly bind to CaMKII promoter and mediate H3K27me3 modification. ( C , D ) ChIP-qPCR of EZH2 occupancy and H3K27me3 binding in the promoter of CaMKII in cardiomyocytes transfected with TINCR siRNA or scrambled siRNA. * P
    Figure Legend Snippet: ( A ) The RIP assay was conducted to confirm whether TINCR could directly bind with EZH2. ( B ) The ChIP assay was performed to verify whether EZH2 could directly bind to CaMKII promoter and mediate H3K27me3 modification. ( C , D ) ChIP-qPCR of EZH2 occupancy and H3K27me3 binding in the promoter of CaMKII in cardiomyocytes transfected with TINCR siRNA or scrambled siRNA. * P

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

    5) Product Images from "Over-expression of oncigenic pesudogene DUXAP10 promotes cell proliferation and invasion by regulating LATS1 and β-catenin in gastric cancer"

    Article Title: Over-expression of oncigenic pesudogene DUXAP10 promotes cell proliferation and invasion by regulating LATS1 and β-catenin in gastric cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0684-8

    DUXAP10 interacts PRC2/LSD1 and HuR to regulate KLF2, LATS1and β-catenin expression. a QPCR was used to examine the levels of potential DUXAP10 targets in GC cells after knockdown of DUXAP10. b KLF2, LATS1and β-catenin protein levels were analyzed by western blot in GC cells after knockdown of DUXAP10. c KLF2 and LATS1 levels were analyzed by qPCR in GC cells after transfection with EZH2, SUZ12 and LSD1 siRNA. d , e ChIP shows SUZ12, EZH2, H3K27me3, LSD1 and H3K4me2 occupancy in the LATS1 and KLF2 promoter region, while knockdown of DUXAP10 decreased their binding ability. f RIP assays show the interaction between HuR and β-catenin mRNA in GC cells, and knockdown of DUXAP10 impaired their interaction ability. * P
    Figure Legend Snippet: DUXAP10 interacts PRC2/LSD1 and HuR to regulate KLF2, LATS1and β-catenin expression. a QPCR was used to examine the levels of potential DUXAP10 targets in GC cells after knockdown of DUXAP10. b KLF2, LATS1and β-catenin protein levels were analyzed by western blot in GC cells after knockdown of DUXAP10. c KLF2 and LATS1 levels were analyzed by qPCR in GC cells after transfection with EZH2, SUZ12 and LSD1 siRNA. d , e ChIP shows SUZ12, EZH2, H3K27me3, LSD1 and H3K4me2 occupancy in the LATS1 and KLF2 promoter region, while knockdown of DUXAP10 decreased their binding ability. f RIP assays show the interaction between HuR and β-catenin mRNA in GC cells, and knockdown of DUXAP10 impaired their interaction ability. * P

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

    6) Product Images from "A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis"

    Article Title: A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

    Journal: The FASEB Journal

    doi: 10.1096/fj.13-241760

    A hypothetical model depicting the central role of G9a- and EZH2-mediated histone methylation and the interdependent and mutually reinforcing crosstalk between histone methylation and DNA methylation in COX-2 epigenetic silencing in IPF. G9a- and EZH2-mediated H3K9me3 and H3K27me3 result in the recruitment of Dnmts and HDAC-containing complexes via HP1 and EZH2/EED, respectively, to the COX-2 promoter, which then leads to or reinforces DNA methylation and histone deacetylation. DNA methylation in turn causes the recruitment of G9a, EZH2, and HDAC-containing complexes through MeCP2 to strengthen H3K9me3, H3K27me3, and histone deacetylation, leading to reinforced epigenetic silencing of the COX-2 gene in IPF. Therefore, G9a- and EZH2-mediated H3K9me3 and H3K27me3 interact with DNA methylation in a bidirectional and mutually dependent manner to reinforce COX-2 epigenetic silencing in IPF. Disruption of any of these epigenetic modifications by inhibition or knockdown of G9a, EZH2, or Dnmt leads to the removal of the other repressive epigenetic modifications, resulting in an active chromatin state and reactivation of COX-2 in IPF.
    Figure Legend Snippet: A hypothetical model depicting the central role of G9a- and EZH2-mediated histone methylation and the interdependent and mutually reinforcing crosstalk between histone methylation and DNA methylation in COX-2 epigenetic silencing in IPF. G9a- and EZH2-mediated H3K9me3 and H3K27me3 result in the recruitment of Dnmts and HDAC-containing complexes via HP1 and EZH2/EED, respectively, to the COX-2 promoter, which then leads to or reinforces DNA methylation and histone deacetylation. DNA methylation in turn causes the recruitment of G9a, EZH2, and HDAC-containing complexes through MeCP2 to strengthen H3K9me3, H3K27me3, and histone deacetylation, leading to reinforced epigenetic silencing of the COX-2 gene in IPF. Therefore, G9a- and EZH2-mediated H3K9me3 and H3K27me3 interact with DNA methylation in a bidirectional and mutually dependent manner to reinforce COX-2 epigenetic silencing in IPF. Disruption of any of these epigenetic modifications by inhibition or knockdown of G9a, EZH2, or Dnmt leads to the removal of the other repressive epigenetic modifications, resulting in an active chromatin state and reactivation of COX-2 in IPF.

    Techniques Used: Methylation, DNA Methylation Assay, Inhibition

    Histone H3 is repressively hypermethylated and HMT recruitment is increased at the COX-2 promoter in F-IPFs. Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K4me3 ( A ), H3K9me3 ( B ), H3K27me3 ( C ), G9a ( D ), SUV39H1 ( E ), EZH2 ( F ), and total histone H3 ( A–C ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A–C ) or to input control ( D–F ). Data are expressed as means ± sem from experiments with 6 separate F-NL and F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: Histone H3 is repressively hypermethylated and HMT recruitment is increased at the COX-2 promoter in F-IPFs. Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K4me3 ( A ), H3K9me3 ( B ), H3K27me3 ( C ), G9a ( D ), SUV39H1 ( E ), EZH2 ( F ), and total histone H3 ( A–C ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A–C ) or to input control ( D–F ). Data are expressed as means ± sem from experiments with 6 separate F-NL and F-IPF cell lines performed in duplicate. * P

    Techniques Used: HMT Assay, Incubation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    Epigenetic inhibitors of G9a, EZH2, and Dnmt1 reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation at the COX-2 promoter in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), RG109 (5 μM), or DZNep (10 nM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), and acetylated histone H3 ( D ) and H4 ( E ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D ), total histone H4 ( E ), or input control ( B ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: Epigenetic inhibitors of G9a, EZH2, and Dnmt1 reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation at the COX-2 promoter in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), RG109 (5 μM), or DZNep (10 nM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), and acetylated histone H3 ( D ) and H4 ( E ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D ), total histone H4 ( E ), or input control ( B ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

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

    G9a and EZH2 siRNAs reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation and H3K4me3 at the COX-2 promoter in F-IPFs. F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the siRNAs for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), acetylated histone H3 ( D ) and H4 ( E ), CBP, p300, PCAF ( F ), and H3K4me3 ( G ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D , G ), total histone H4 ( E ), or input control ( B , F ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: G9a and EZH2 siRNAs reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation and H3K4me3 at the COX-2 promoter in F-IPFs. F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the siRNAs for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), acetylated histone H3 ( D ) and H4 ( E ), CBP, p300, PCAF ( F ), and H3K4me3 ( G ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D , G ), total histone H4 ( E ), or input control ( B , F ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

    Techniques Used: Transfection, Incubation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    COX-2 promoter DNA methylation and Dnmt association with the COX-2 promoter are increased in F-IPFs. A ) Confluent F-NLs and F-IPFs were serum starved for 24 h and then lysed. DNA was extracted. Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for different regions of the COX-2 promoter and its upstream and downstream regions. B–D ) Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. Dnmt1 ( B ), Dnmt3a ( C ), and MeCP2 ( D ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. E , F ) Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. MeCP2 was immunoprecipitated with specific antibody first, and then the IPs were immunoprecipitated again with antibodies against G9a, EZH2, Dnmt1, and Dnmt3a ( E ) and NCoR, CoREST, and mSin3a ( F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-NL and/or F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: COX-2 promoter DNA methylation and Dnmt association with the COX-2 promoter are increased in F-IPFs. A ) Confluent F-NLs and F-IPFs were serum starved for 24 h and then lysed. DNA was extracted. Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for different regions of the COX-2 promoter and its upstream and downstream regions. B–D ) Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. Dnmt1 ( B ), Dnmt3a ( C ), and MeCP2 ( D ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. E , F ) Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. MeCP2 was immunoprecipitated with specific antibody first, and then the IPs were immunoprecipitated again with antibodies against G9a, EZH2, Dnmt1, and Dnmt3a ( E ) and NCoR, CoREST, and mSin3a ( F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-NL and/or F-IPF cell lines performed in duplicate. * P

    Techniques Used: DNA Methylation Assay, Methylation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Incubation, Sonication

    HP1, PRC1, and repressive epigenetic enzymes are associated with the COX-2 promoter in F-IPFs. Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), H3K27me3 ( B ), HP1 ( C and D ), EZH2 ( E ), and EED ( F ) were immunoprecipitated with specific antibodies first, and then the IPs were immunoprecipitated again with antibodies against HP1 ( A ), PRC1 ( B ), G9a ( C ), Dnmt1, Dnmt3a ( C and E ), EED ( E ), and NCoR, CoREST, and mSin3a ( D , F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate.
    Figure Legend Snippet: HP1, PRC1, and repressive epigenetic enzymes are associated with the COX-2 promoter in F-IPFs. Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), H3K27me3 ( B ), HP1 ( C and D ), EZH2 ( E ), and EED ( F ) were immunoprecipitated with specific antibodies first, and then the IPs were immunoprecipitated again with antibodies against HP1 ( A ), PRC1 ( B ), G9a ( C ), Dnmt1, Dnmt3a ( C and E ), EED ( E ), and NCoR, CoREST, and mSin3a ( D , F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate.

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

    Epigenetic inhibitors of G9a, EZH2, and Dnmt1 restore COX-2 expression and PGE 2 production in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), DZNep (10 nM), or RG108 (5 μM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h ( A ) or 24 h ( B , C ). A ) Total RNA was isolated, and mRNA levels of COX-2 and the internal control β 2 -microglobulin (β2M) were determined by real-time RT-PCR. The data are calculated as the ratio of COX-2 mRNA and β2M mRNA and are expressed as means ± sem of 6 separate experiments performed in duplicate. B ) Total cell lysates were collected for Western blotting analysis of COX-2 with GAPDH as the loading control. Data are representative of 3 separate experiments with different F-IPF cell lines. Relative density was calculated by normalizing the density of the COX-2 bands against that of the GAPDH bands from 3 separate experiments. C ) Culture media were collected for PGE 2 assay. For panels A , C , data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: Epigenetic inhibitors of G9a, EZH2, and Dnmt1 restore COX-2 expression and PGE 2 production in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), DZNep (10 nM), or RG108 (5 μM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h ( A ) or 24 h ( B , C ). A ) Total RNA was isolated, and mRNA levels of COX-2 and the internal control β 2 -microglobulin (β2M) were determined by real-time RT-PCR. The data are calculated as the ratio of COX-2 mRNA and β2M mRNA and are expressed as means ± sem of 6 separate experiments performed in duplicate. B ) Total cell lysates were collected for Western blotting analysis of COX-2 with GAPDH as the loading control. Data are representative of 3 separate experiments with different F-IPF cell lines. Relative density was calculated by normalizing the density of the COX-2 bands against that of the GAPDH bands from 3 separate experiments. C ) Culture media were collected for PGE 2 assay. For panels A , C , data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

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

    G9a and EZH2 siRNAs restore COX-2 expression and PGE 2 production in F-IPFs. A , B ) F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d. Total RNA was isolated, and mRNA levels of G9a ( A ) and EZH2 ( B ) were determined by real-time RT-PCR. The data are calculated as the ratio of G9a and EZH2 mRNA and the internal control β 2 -microglobulin (β2M) mRNA. C−F ) F-IPFs transfected with or without siRNAs were serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) for a further 4 h ( C ) or 24 h ( D−F ). C ) Total RNA was isolated, and mRNA levels of COX-2 were determined by real-time RT-PCR. The data are calculated as the ratio of COX-2 mRNA and the internal control β2M mRNA. D ) Total cell lysates were collected for Western blotting analysis of COX-2, G9a, and EZH2 with β2M as the loading control. Data are representative of 3 separate experiments with different F-IPF cell lines. E ) Relative density of the Western blot was calculated by normalizing the density of the COX-2, EZH2, and G9a bands against that of the β2M bands from 3 separate experiments. F ) Culture media were collected for PGE 2 assay. For panels A−C and F , data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: G9a and EZH2 siRNAs restore COX-2 expression and PGE 2 production in F-IPFs. A , B ) F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d. Total RNA was isolated, and mRNA levels of G9a ( A ) and EZH2 ( B ) were determined by real-time RT-PCR. The data are calculated as the ratio of G9a and EZH2 mRNA and the internal control β 2 -microglobulin (β2M) mRNA. C−F ) F-IPFs transfected with or without siRNAs were serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) for a further 4 h ( C ) or 24 h ( D−F ). C ) Total RNA was isolated, and mRNA levels of COX-2 were determined by real-time RT-PCR. The data are calculated as the ratio of COX-2 mRNA and the internal control β2M mRNA. D ) Total cell lysates were collected for Western blotting analysis of COX-2, G9a, and EZH2 with β2M as the loading control. Data are representative of 3 separate experiments with different F-IPF cell lines. E ) Relative density of the Western blot was calculated by normalizing the density of the COX-2, EZH2, and G9a bands against that of the β2M bands from 3 separate experiments. F ) Culture media were collected for PGE 2 assay. For panels A−C and F , data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

    Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR, Incubation, Western Blot

    G9a and EZH2 inhibitors and siRNAs reduce COX-2 promoter DNA methylation in F-IPFs. A , B ) F-IPFs were incubated without or with BIX-01294 (100 nM), DZNep (10 nM), or RG109 (5 μM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d. Cells were then lysed, and DNA was extracted and sheared. A ) Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for the COX-2 promoter DNA. B ) The protein-DNA complexes were cross-linked by formaldehyde treatment and chromatin pellets were extracted and sonicated. MeCP2 was immunoprecipitated with a specific antibody. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to the input control. C ) F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d. The cells were then lysed, and DNA was extracted and sheared. Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for the COX-2 promoter DNA with samples from F-NLs as a reference. Data are expressed as means ± sem from experiments with 6 separate F-IPF and F-NL cell lines performed in duplicate. * P
    Figure Legend Snippet: G9a and EZH2 inhibitors and siRNAs reduce COX-2 promoter DNA methylation in F-IPFs. A , B ) F-IPFs were incubated without or with BIX-01294 (100 nM), DZNep (10 nM), or RG109 (5 μM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d. Cells were then lysed, and DNA was extracted and sheared. A ) Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for the COX-2 promoter DNA. B ) The protein-DNA complexes were cross-linked by formaldehyde treatment and chromatin pellets were extracted and sonicated. MeCP2 was immunoprecipitated with a specific antibody. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to the input control. C ) F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d. The cells were then lysed, and DNA was extracted and sheared. Methylated DNA was immunoprecipitated with an antibody against 5-methylcytosine. The associated DNA was amplified by real-time PCR using specific primers for the COX-2 promoter DNA with samples from F-NLs as a reference. Data are expressed as means ± sem from experiments with 6 separate F-IPF and F-NL cell lines performed in duplicate. * P

    Techniques Used: DNA Methylation Assay, Incubation, Methylation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Sonication, Transfection

    7) Product Images from "Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development"

    Article Title: Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003909

    Ezh2 inactivation in mesenchymal progenitor, osteoblast, and chondrocyte lineages. A , overview of mesenchymal lineage and mouse Cre drivers. Lineage-specific conditional inactivation of Ezh2 using the mesenchymal progenitor driver Prrx1-Cre , the osteoprogenitor driver Osx1 -Cre, and the chondroprogenitor driver Col2a1-Cre was performed. B , Alcian blue/eosin analysis of proximal tibiae from WT and cKO mice at adolescence. C , average depth, growth plate depth, and hypertrophic zone depths within the growth plate were determined using ImageJ software. D , comparison of epiphysis and growth plate depth in WT and cKO animals. *, p
    Figure Legend Snippet: Ezh2 inactivation in mesenchymal progenitor, osteoblast, and chondrocyte lineages. A , overview of mesenchymal lineage and mouse Cre drivers. Lineage-specific conditional inactivation of Ezh2 using the mesenchymal progenitor driver Prrx1-Cre , the osteoprogenitor driver Osx1 -Cre, and the chondroprogenitor driver Col2a1-Cre was performed. B , Alcian blue/eosin analysis of proximal tibiae from WT and cKO mice at adolescence. C , average depth, growth plate depth, and hypertrophic zone depths within the growth plate were determined using ImageJ software. D , comparison of epiphysis and growth plate depth in WT and cKO animals. *, p

    Techniques Used: Mouse Assay, Software

    Chondrogenic genes exhibit normal expression upon loss of Ezh2 and H3K27me3. A , IMCs were isolated from WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 mice and plated in micromass culture for 14 days ( D ) in the presence of chondrogenic mixture ( Chondro mix ). B , abundance of Ezh2 and H3K27me3 was reduced in cKO Col2 micromasses compared with the WT. The numbers on the right indicate locations of molecular weight markers (kilodalton). C , histological staining of WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 micromasses show a reduced Alcian blue extracellular matrix in cKO Col2 chondrocytes. D , RNA-Seq analysis was performed on WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 IMCs on day 3, day 7, and day 14 of culture. Gene expression data were filtered to exclude genes that were not expressed in any of the samples (0 RPKM, 19,641 of 23,359 genes were visualized). Hierarchical clustering of the filtered RNA-Seq identified three clusters that grouped together WT and cKO Col2 samples taken at similar time points, indicating that the majority of expressed genes were similar between samples. E , chondrogenic genes associated with immature and proliferating chondrocytes ( Col2a1 , Acan , and Sox9 ) are highly expressed on day 3, whereas the hypertrophic markers Col10a1 and Vegfa are up-regulated on day 14. F , validation of RNA-Seq data shows good concordance with quantitative PCR.
    Figure Legend Snippet: Chondrogenic genes exhibit normal expression upon loss of Ezh2 and H3K27me3. A , IMCs were isolated from WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 mice and plated in micromass culture for 14 days ( D ) in the presence of chondrogenic mixture ( Chondro mix ). B , abundance of Ezh2 and H3K27me3 was reduced in cKO Col2 micromasses compared with the WT. The numbers on the right indicate locations of molecular weight markers (kilodalton). C , histological staining of WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 micromasses show a reduced Alcian blue extracellular matrix in cKO Col2 chondrocytes. D , RNA-Seq analysis was performed on WT ( Ezh2 f/f ; Col2a1-Cre −/− ) and cKO Col2 IMCs on day 3, day 7, and day 14 of culture. Gene expression data were filtered to exclude genes that were not expressed in any of the samples (0 RPKM, 19,641 of 23,359 genes were visualized). Hierarchical clustering of the filtered RNA-Seq identified three clusters that grouped together WT and cKO Col2 samples taken at similar time points, indicating that the majority of expressed genes were similar between samples. E , chondrogenic genes associated with immature and proliferating chondrocytes ( Col2a1 , Acan , and Sox9 ) are highly expressed on day 3, whereas the hypertrophic markers Col10a1 and Vegfa are up-regulated on day 14. F , validation of RNA-Seq data shows good concordance with quantitative PCR.

    Techniques Used: Expressing, Isolation, Mouse Assay, Molecular Weight, Staining, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Differential gene expression analysis shows up-regulation of osteogenic genes in Ezh2 -deficient immature chondrocyte cultures. A , differential gene expression analysis of RNA-Seq from WT ( Ezh2 wt/wt ; Col2a1-Cre +/− ) and cKO Col2 immature chondrocytes revealed 57 genes that were commonly up-regulated more than 1.4-fold in Ezh2 -deficient cultures on days 3, 7, and 14. The analysis was limited to genes with a minimal expression of 0.1 RPKM in the cKO group at each time point. B , the osteogenic extracellular matrix genes Ibsp , Bglap , and Wnt16 were up-regulated in cKO Col2 chondrocytes as well as some nonosteogenic genes such as Cyp27b1 , Ebf2 , and Otor .
    Figure Legend Snippet: Differential gene expression analysis shows up-regulation of osteogenic genes in Ezh2 -deficient immature chondrocyte cultures. A , differential gene expression analysis of RNA-Seq from WT ( Ezh2 wt/wt ; Col2a1-Cre +/− ) and cKO Col2 immature chondrocytes revealed 57 genes that were commonly up-regulated more than 1.4-fold in Ezh2 -deficient cultures on days 3, 7, and 14. The analysis was limited to genes with a minimal expression of 0.1 RPKM in the cKO group at each time point. B , the osteogenic extracellular matrix genes Ibsp , Bglap , and Wnt16 were up-regulated in cKO Col2 chondrocytes as well as some nonosteogenic genes such as Cyp27b1 , Ebf2 , and Otor .

    Techniques Used: Expressing, RNA Sequencing Assay

    Ezh2-deficient adolescent mice exhibit reduced bone density that is restored during adulthood. A , representative micro-CT 3D reconstruction of the proximal tibial metaphysis of WT and cKO Col2 mice. B , BV/TV and Tb.Th measured in the proximal tibial metaphysis were lower in Ezh2 -deficient mice at 4 weeks of age ( n = 3/group), although, upon skeletal maturity (8 weeks), no differences were observed ( n = 3/group). Further, these parameters were also unchanged at 24 weeks of age when measured in the lumbar vertebrae (WT, n = 3; cKO, n = 4). *, p
    Figure Legend Snippet: Ezh2-deficient adolescent mice exhibit reduced bone density that is restored during adulthood. A , representative micro-CT 3D reconstruction of the proximal tibial metaphysis of WT and cKO Col2 mice. B , BV/TV and Tb.Th measured in the proximal tibial metaphysis were lower in Ezh2 -deficient mice at 4 weeks of age ( n = 3/group), although, upon skeletal maturity (8 weeks), no differences were observed ( n = 3/group). Further, these parameters were also unchanged at 24 weeks of age when measured in the lumbar vertebrae (WT, n = 3; cKO, n = 4). *, p

    Techniques Used: Mouse Assay, Micro-CT

    Cartilage-specific deletion of Ezh2 does not affect skeletal developmental. A , immunohistochemical analysis of H3K27me3 and Ezh2 abundance in proximal tibiae from post-natal day 1 WT and cKO Col2 mice. B , Alcian blue/alizarin red whole-mount staining did not reveal growth abnormalities in post-natal day 1 mice ( left panels ), and radiographic analysis was similar between WT and cKO Col2 mice at 4 weeks ( center panels ) and 8 weeks of age ( right panels ). C , body weight was used as an indicator for altered growth. On post-natal day 1, adolescence, and adulthood, the body weights were similar between genotypes.
    Figure Legend Snippet: Cartilage-specific deletion of Ezh2 does not affect skeletal developmental. A , immunohistochemical analysis of H3K27me3 and Ezh2 abundance in proximal tibiae from post-natal day 1 WT and cKO Col2 mice. B , Alcian blue/alizarin red whole-mount staining did not reveal growth abnormalities in post-natal day 1 mice ( left panels ), and radiographic analysis was similar between WT and cKO Col2 mice at 4 weeks ( center panels ) and 8 weeks of age ( right panels ). C , body weight was used as an indicator for altered growth. On post-natal day 1, adolescence, and adulthood, the body weights were similar between genotypes.

    Techniques Used: Immunohistochemistry, Mouse Assay, Staining

    Ezh2-deficient chondrocytes exhibit enhanced expression of an osteogenic-program in vitro , but genetic inactivation does not result in osteoarthritis in vivo . A , expression of chondrogenic, osteogenic, cell–cell contact/extracellular matrix, and other genes by RNA-Seq. Ezh2 -deficient IMCs exhibit up-regulation of osteogenic genes on day 14 compared with WT ( Ezh2 wt/wt ; Col2a1-Cre +/− ) IMC cultures. B , osteoblast-associated genes, including Sp7(Osx ) and Bmp2 , the osteocyte-associated extracellular matrix gene Mepe , as well as the WNT pathway inhibitors Sost and Dkk1 are up-regulated in cKO Col2 IMC cultures. C , protein levels of the transcription factor Sp7(Osx ) were also enhanced in Ezh2 -deficient IMC cultures. The numbers on the right indicate the location of molecular weight markers (kilodalton). D , WT and cKO Col2 mice were aged to 24 weeks, and micro-CT analysis was performed on the right knee joint. No changes in the subchondral bone were observed ( n = 4/group).
    Figure Legend Snippet: Ezh2-deficient chondrocytes exhibit enhanced expression of an osteogenic-program in vitro , but genetic inactivation does not result in osteoarthritis in vivo . A , expression of chondrogenic, osteogenic, cell–cell contact/extracellular matrix, and other genes by RNA-Seq. Ezh2 -deficient IMCs exhibit up-regulation of osteogenic genes on day 14 compared with WT ( Ezh2 wt/wt ; Col2a1-Cre +/− ) IMC cultures. B , osteoblast-associated genes, including Sp7(Osx ) and Bmp2 , the osteocyte-associated extracellular matrix gene Mepe , as well as the WNT pathway inhibitors Sost and Dkk1 are up-regulated in cKO Col2 IMC cultures. C , protein levels of the transcription factor Sp7(Osx ) were also enhanced in Ezh2 -deficient IMC cultures. The numbers on the right indicate the location of molecular weight markers (kilodalton). D , WT and cKO Col2 mice were aged to 24 weeks, and micro-CT analysis was performed on the right knee joint. No changes in the subchondral bone were observed ( n = 4/group).

    Techniques Used: Expressing, In Vitro, In Vivo, RNA Sequencing Assay, Molecular Weight, Mouse Assay, Micro-CT

    8) Product Images from "Exosome-transmitted lncRNA UFC1 promotes non-small-cell lung cancer progression by EZH2-mediated epigenetic silencing of PTEN expression"

    Article Title: Exosome-transmitted lncRNA UFC1 promotes non-small-cell lung cancer progression by EZH2-mediated epigenetic silencing of PTEN expression

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2409-0

    Proposed model for the oncogenic roles of UFC1 in NSCLC. Exosome-transmitted UFC1 epigenetically silences PTEN expression via binding to EZH2, which promotes Akt pathway activation and increases cell proliferation, migration and invasion in NSCLC.
    Figure Legend Snippet: Proposed model for the oncogenic roles of UFC1 in NSCLC. Exosome-transmitted UFC1 epigenetically silences PTEN expression via binding to EZH2, which promotes Akt pathway activation and increases cell proliferation, migration and invasion in NSCLC.

    Techniques Used: Expressing, Binding Assay, Activation Assay, Migration

    UFC1 interacts with EZH2 to suppress PTEN expression and activate Akt pathway. a QRT-PCR analysis of UFC1 expression levels in different subcellular fractions of A549 cells. b RIP assays with antibodies against EZH2 and IgG control and A549 cell extracts. RNA levels in the immunoprecipitates were detected by qRT-PCR. Expression levels of UFC1 are presented as fold enrichment to input. c QRT-PCR analysis of PTEN expression in NSCLC cells with UFC1 knockdown or overexpression. d Western blot analysis of PTEN and p-Akt expression in NSCLC cells with UFC1 knockdown or overexpression. e ChIP-PCR analysis of EZH2 occupancy and H3K27me3 binding in the PTEN promoter in A549 cells transfected with sh-UFC1 and sh-Ctrl. f QRT-PCR and western blot analysis of PTEN expression in A549 cells with EZH2 knockdown. g Correlation analysis of UFC1 and PTEN expression levels in tumor tissues of NSCLC patients. The experiments were repeated for three times. * P
    Figure Legend Snippet: UFC1 interacts with EZH2 to suppress PTEN expression and activate Akt pathway. a QRT-PCR analysis of UFC1 expression levels in different subcellular fractions of A549 cells. b RIP assays with antibodies against EZH2 and IgG control and A549 cell extracts. RNA levels in the immunoprecipitates were detected by qRT-PCR. Expression levels of UFC1 are presented as fold enrichment to input. c QRT-PCR analysis of PTEN expression in NSCLC cells with UFC1 knockdown or overexpression. d Western blot analysis of PTEN and p-Akt expression in NSCLC cells with UFC1 knockdown or overexpression. e ChIP-PCR analysis of EZH2 occupancy and H3K27me3 binding in the PTEN promoter in A549 cells transfected with sh-UFC1 and sh-Ctrl. f QRT-PCR and western blot analysis of PTEN expression in A549 cells with EZH2 knockdown. g Correlation analysis of UFC1 and PTEN expression levels in tumor tissues of NSCLC patients. The experiments were repeated for three times. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Over Expression, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Transfection

    9) Product Images from "Long noncoding RNA HOXA-AS2 promotes gastric cancer proliferation by epigenetically silencing P21/PLK3/DDIT3 expression"

    Article Title: Long noncoding RNA HOXA-AS2 promotes gastric cancer proliferation by epigenetically silencing P21/PLK3/DDIT3 expression

    Journal: Oncotarget

    doi:

    HOXA-AS2 epigenetically silences P21/PLK3/DDIT3 transcription by binding with EZH2 A. HOXA-AS2 expression levels in cell nucleus or cytoplasm of BGC-823, SGC-7901 and AGS cells were detected by qRT-PCR. U6 was used as a nucleus marker and GAPDH was used as a cytosol marker. B. Gene set containing EZH2-repressed genes were significantly enriched in genes upregulated by HOXA-AS2 knockdown. C. RIP experiments were performed in BGC-823, SGC-7901, AGS cells and the coprecipitated RNA was subjected to qRT-PCR for HOXA-AS2. The fold enrichment of HOXA-AS2 in EZH2/SUZ12/DNMT1 RIP is relative to its matching IgG control. D. , E. and F. ChIp-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the P21/PLK3/DDIT3 promoters in BGC-823, SGC-7901, AGS cells treated with si-HOXA-AS2 (48h) or si-NC; IgG as a negative control. Error bars indicate mean ± standard errors of the mean. * P
    Figure Legend Snippet: HOXA-AS2 epigenetically silences P21/PLK3/DDIT3 transcription by binding with EZH2 A. HOXA-AS2 expression levels in cell nucleus or cytoplasm of BGC-823, SGC-7901 and AGS cells were detected by qRT-PCR. U6 was used as a nucleus marker and GAPDH was used as a cytosol marker. B. Gene set containing EZH2-repressed genes were significantly enriched in genes upregulated by HOXA-AS2 knockdown. C. RIP experiments were performed in BGC-823, SGC-7901, AGS cells and the coprecipitated RNA was subjected to qRT-PCR for HOXA-AS2. The fold enrichment of HOXA-AS2 in EZH2/SUZ12/DNMT1 RIP is relative to its matching IgG control. D. , E. and F. ChIp-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the P21/PLK3/DDIT3 promoters in BGC-823, SGC-7901, AGS cells treated with si-HOXA-AS2 (48h) or si-NC; IgG as a negative control. Error bars indicate mean ± standard errors of the mean. * P

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

    HOXA-AS2 knockdown increases the expression of genes involved in cell proliferation A. , B. HOXA-AS2 had significantly negative correlation with genes involved in P53 signaling pathway and the genes targeted by EZH2 in GC dataset (GSE15459). C. QRT-PCR was used to detect P21 mRNA levels after HOXA-AS2 knockdown in BGC-823, SGC-7901 and AGS cells. D. Gene Ontology analysis for all genes with altered expressions between si-NC and si-HOXA-AS2 treated BGC-823 cells in vitro . E. GSEA analysis indicated that cell proliferation process gene set were significantly enriched in genes upregulated by HOXA-AS2 knockdown. F. QRT-PCR was used to validate the changes of several mRNAs involved in cell proliferation. Error bars indicate mean ± standard errors of the mean. * P
    Figure Legend Snippet: HOXA-AS2 knockdown increases the expression of genes involved in cell proliferation A. , B. HOXA-AS2 had significantly negative correlation with genes involved in P53 signaling pathway and the genes targeted by EZH2 in GC dataset (GSE15459). C. QRT-PCR was used to detect P21 mRNA levels after HOXA-AS2 knockdown in BGC-823, SGC-7901 and AGS cells. D. Gene Ontology analysis for all genes with altered expressions between si-NC and si-HOXA-AS2 treated BGC-823 cells in vitro . E. GSEA analysis indicated that cell proliferation process gene set were significantly enriched in genes upregulated by HOXA-AS2 knockdown. F. QRT-PCR was used to validate the changes of several mRNAs involved in cell proliferation. Error bars indicate mean ± standard errors of the mean. * P

    Techniques Used: Expressing, Quantitative RT-PCR, In Vitro

    10) Product Images from "Long noncoding RNA ZFAS1 promotes gastric cancer cells proliferation by epigenetically repressing KLF2 and NKD2 expression"

    Article Title: Long noncoding RNA ZFAS1 promotes gastric cancer cells proliferation by epigenetically repressing KLF2 and NKD2 expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9611

    ZFAS1 interacted with EZH2 and LSD1/CoRET, and regulate KLF2 and NKD2 expression A . Relative ZFAS1 levels in BGC823 and SGC7901 cell cytoplasm or Nucleus were detected by qPCR. GAPDH was used as cytoplasm control and U1 was used as nuclear control. B . ZFAS1RNA levels in immunoprecipitates with EZH2, SUZ12, LSD1 et al. antibodies were determined by qPCR. The expression levels of ZFAS1 RNA were presented as fold enrichment relative to IgG. C . SNRNP70 RNA levels in immunoprecipitates with U1 antibodies were used as positive control. D . EZH2 and LSD1 protein levels in immunoprecipitates with ZFAS1 RNA were determined by western blot. HuR protein immunoprecipitates with AR RNA was used as positive control. E . Pathways analysis of ZFAS1 associated genes in gastric cancer tissues. F . The levels of KLF2, LATS1/2, P21, P15, and NKD2 et al. mRNA were determined by qPCR when knockdown of ZFAS1 in BGC823 and SGC7901 cells. G . The KLF2 and NKD2 protein levels were determined by western blot in ZFAS1 knockdown BGC823 and SGC7901 cells. All experiments were performed in biological triplicates. *P
    Figure Legend Snippet: ZFAS1 interacted with EZH2 and LSD1/CoRET, and regulate KLF2 and NKD2 expression A . Relative ZFAS1 levels in BGC823 and SGC7901 cell cytoplasm or Nucleus were detected by qPCR. GAPDH was used as cytoplasm control and U1 was used as nuclear control. B . ZFAS1RNA levels in immunoprecipitates with EZH2, SUZ12, LSD1 et al. antibodies were determined by qPCR. The expression levels of ZFAS1 RNA were presented as fold enrichment relative to IgG. C . SNRNP70 RNA levels in immunoprecipitates with U1 antibodies were used as positive control. D . EZH2 and LSD1 protein levels in immunoprecipitates with ZFAS1 RNA were determined by western blot. HuR protein immunoprecipitates with AR RNA was used as positive control. E . Pathways analysis of ZFAS1 associated genes in gastric cancer tissues. F . The levels of KLF2, LATS1/2, P21, P15, and NKD2 et al. mRNA were determined by qPCR when knockdown of ZFAS1 in BGC823 and SGC7901 cells. G . The KLF2 and NKD2 protein levels were determined by western blot in ZFAS1 knockdown BGC823 and SGC7901 cells. All experiments were performed in biological triplicates. *P

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

    ZFAS1 recruits EZH2/ LSD1 to KLF2 and NKD2 promoter and represses their transcription A . The EZH2 and LSD1 expression levels were detected by qPCR when knockdown of EZH2 or LSD1 in BGC823 and SGC7901 cells. B . The KLF2 and NKD2 expression levels were detected by qPCR when knockdown of EZH2 or LSD1 in BGC823 and SGC7901 cells. C . ChIP–qPCR analysis of LSD1and EZH2 occupancy, H3K4me2 and H3K27me3 binding in the KLF2 or NKD2 promoter in BGC823 and SGC7901 cells, and IgG as a negative control. D . ChIP–qPCR analysis of LSD1 occupancy in the PSA, and EZH2 occupancy in the MYT-1 promoter in BGC823 and SGC7901 cells, which was used as positive control. E . ChIP–qPCR analysis of LSD1and EZH2 occupancy, H3K4me2 and H3K27me3 binding in the KLF2 or NKD2 promoter after knockdown of ZFAS1. All experiments were performed in biological triplicates.*P
    Figure Legend Snippet: ZFAS1 recruits EZH2/ LSD1 to KLF2 and NKD2 promoter and represses their transcription A . The EZH2 and LSD1 expression levels were detected by qPCR when knockdown of EZH2 or LSD1 in BGC823 and SGC7901 cells. B . The KLF2 and NKD2 expression levels were detected by qPCR when knockdown of EZH2 or LSD1 in BGC823 and SGC7901 cells. C . ChIP–qPCR analysis of LSD1and EZH2 occupancy, H3K4me2 and H3K27me3 binding in the KLF2 or NKD2 promoter in BGC823 and SGC7901 cells, and IgG as a negative control. D . ChIP–qPCR analysis of LSD1 occupancy in the PSA, and EZH2 occupancy in the MYT-1 promoter in BGC823 and SGC7901 cells, which was used as positive control. E . ChIP–qPCR analysis of LSD1and EZH2 occupancy, H3K4me2 and H3K27me3 binding in the KLF2 or NKD2 promoter after knockdown of ZFAS1. All experiments were performed in biological triplicates.*P

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

    11) Product Images from "LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer"

    Article Title: LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.150

    The association of HOTAIR with the PRC2 protein complex is critical for its regulation of miR34a . ( a ) Genome browser and analyzed H3K27me3 enrichment peaks in the miR34a promoter region. ( b ) RIP experiments were performed using the EZH2 antibodies for immunoprecipitation. Specific primers for HOTAIR were used to detect HOTAIR . ( c and d ) Expression of miR34a in BGC-823, SGC-7901 and MGC-803 cells transfected with si-EZH2, si-SUZ12 was detected by qRT-PCR. ( e and f ) ChIP analyses in SGC-7901 transfected with Si-HOTAIR and Si-EZH2 cells were performed on the miR34a promoter regions using anti-H3K27me3 and EZH2 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates. * P
    Figure Legend Snippet: The association of HOTAIR with the PRC2 protein complex is critical for its regulation of miR34a . ( a ) Genome browser and analyzed H3K27me3 enrichment peaks in the miR34a promoter region. ( b ) RIP experiments were performed using the EZH2 antibodies for immunoprecipitation. Specific primers for HOTAIR were used to detect HOTAIR . ( c and d ) Expression of miR34a in BGC-823, SGC-7901 and MGC-803 cells transfected with si-EZH2, si-SUZ12 was detected by qRT-PCR. ( e and f ) ChIP analyses in SGC-7901 transfected with Si-HOTAIR and Si-EZH2 cells were performed on the miR34a promoter regions using anti-H3K27me3 and EZH2 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates. * P

    Techniques Used: Immunoprecipitation, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation

    12) Product Images from "Upregulated long non-coding RNA AGAP2-AS1 represses LATS2 and KLF2 expression through interacting with EZH2 and LSD1 in non-small-cell lung cancer cells"

    Article Title: Upregulated long non-coding RNA AGAP2-AS1 represses LATS2 and KLF2 expression through interacting with EZH2 and LSD1 in non-small-cell lung cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.126

    AGAP2-AS1 interacted with EZH2 and LSD1 to repress KLF2 and LATS2 expression. ( a ) Relative AGAP2-AS1 levels in cell cytoplasm or nucleus of H1299 and H1975 cells were detected by qPCR. GAPDH was used as cytoplasm control and U1 was used as nucleus control. The distribution of AGAP2-AS1 RNA in the cytoplasm or the nucleus was presented as percentage rate of total RNA. ( b ) RNA levels in immunoprecipitates with EZH2, SUZ12, LSD1, CoREST, SIRT1 and TDP43 were determined by qPCR. Expression levels of AGAP2-AS1 RNA were presented as fold enrichment relative to IgG immunoprecipitate. ( c ) The levels of P15, P21, LATS1, LATS2, RND1, KLF2, E-cadherin, PTEN, RRAD and ASPP2 messenger RNA were determined by qPCR when knockdown of AGAP2-AS1 in H1299 and H1975 cells. ( d ) The KLF2 and LATS2 protein levels were determined by western blot analysis in AGAP2-AS1 knockdown H1299 and H1975 cells. ( e ) The KLF2 and LATS2 expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in H1299 and H1975 cells. ( f and g ) Chromatin immunoprecipitation-qPCR analysis of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding to the KLF2 or LATS2 promoter regions in H1299 and H1975 cells, and IgG as a negative control. The mean values and S.E.M. were calculated from triplicates of a representative experiment. * P
    Figure Legend Snippet: AGAP2-AS1 interacted with EZH2 and LSD1 to repress KLF2 and LATS2 expression. ( a ) Relative AGAP2-AS1 levels in cell cytoplasm or nucleus of H1299 and H1975 cells were detected by qPCR. GAPDH was used as cytoplasm control and U1 was used as nucleus control. The distribution of AGAP2-AS1 RNA in the cytoplasm or the nucleus was presented as percentage rate of total RNA. ( b ) RNA levels in immunoprecipitates with EZH2, SUZ12, LSD1, CoREST, SIRT1 and TDP43 were determined by qPCR. Expression levels of AGAP2-AS1 RNA were presented as fold enrichment relative to IgG immunoprecipitate. ( c ) The levels of P15, P21, LATS1, LATS2, RND1, KLF2, E-cadherin, PTEN, RRAD and ASPP2 messenger RNA were determined by qPCR when knockdown of AGAP2-AS1 in H1299 and H1975 cells. ( d ) The KLF2 and LATS2 protein levels were determined by western blot analysis in AGAP2-AS1 knockdown H1299 and H1975 cells. ( e ) The KLF2 and LATS2 expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in H1299 and H1975 cells. ( f and g ) Chromatin immunoprecipitation-qPCR analysis of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding to the KLF2 or LATS2 promoter regions in H1299 and H1975 cells, and IgG as a negative control. The mean values and S.E.M. were calculated from triplicates of a representative experiment. * P

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

    13) Product Images from "A novel lncRNA, LL22NC03-N64E9.1, represses KLF2 transcription through binding with EZH2 in colorectal cancer"

    Article Title: A novel lncRNA, LL22NC03-N64E9.1, represses KLF2 transcription through binding with EZH2 in colorectal cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19738

    LL22NC03-N64E9.1 epigenetically silences KLF2 transcription by binding with EZH2 (A) Relative LL22NC03-N64E9.1 levels in cell cytoplasm or nucleus of SW480 cell lines were detected by qRT-PCR. (B) Heat map of altered genes in LL22NC03-N64E9.1 knockdown SW480 cells compared with control cells. (C) The levels of KLF2, p21, PTEN, Trail, p15, p53, Bax and Bcl-2 mRNA were detected by qRT-PCR when knockdown of LL22NC03-N64E9.1 in SW480 cells. (D) RIP assays were performed in SW480 cells and the coprecipitated RNA was subjected to qRT-PCR for LL22NC03-N64E9.1. (E) The expression of EZH2, KLF2, p21, PTEN, Trail and p15 in SW480 cells, after knockdown of EZH2. (F) The KLF2 protein levels were determined by western blot in LL22NC03-N64E9.1 or EZH2 knockdown SW480 cells. (G, H) ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the KLF2 locus after siRNA treatment targeting si-NC or si-LL22NC03-N64E9.1 in SW480 cells, (I) The relationship between LL22NC03-N64E9.1 expression and KLF2 mRNA levels was analyzed in the profile of CRC patient tissue from Gene Expression Omnibus (GEO). Representative images and data based on three independent experiments. Bars: s.d, *P
    Figure Legend Snippet: LL22NC03-N64E9.1 epigenetically silences KLF2 transcription by binding with EZH2 (A) Relative LL22NC03-N64E9.1 levels in cell cytoplasm or nucleus of SW480 cell lines were detected by qRT-PCR. (B) Heat map of altered genes in LL22NC03-N64E9.1 knockdown SW480 cells compared with control cells. (C) The levels of KLF2, p21, PTEN, Trail, p15, p53, Bax and Bcl-2 mRNA were detected by qRT-PCR when knockdown of LL22NC03-N64E9.1 in SW480 cells. (D) RIP assays were performed in SW480 cells and the coprecipitated RNA was subjected to qRT-PCR for LL22NC03-N64E9.1. (E) The expression of EZH2, KLF2, p21, PTEN, Trail and p15 in SW480 cells, after knockdown of EZH2. (F) The KLF2 protein levels were determined by western blot in LL22NC03-N64E9.1 or EZH2 knockdown SW480 cells. (G, H) ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the KLF2 locus after siRNA treatment targeting si-NC or si-LL22NC03-N64E9.1 in SW480 cells, (I) The relationship between LL22NC03-N64E9.1 expression and KLF2 mRNA levels was analyzed in the profile of CRC patient tissue from Gene Expression Omnibus (GEO). Representative images and data based on three independent experiments. Bars: s.d, *P

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

    14) Product Images from "Over-expression of oncigenic pesudogene DUXAP10 promotes cell proliferation and invasion by regulating LATS1 and β-catenin in gastric cancer"

    Article Title: Over-expression of oncigenic pesudogene DUXAP10 promotes cell proliferation and invasion by regulating LATS1 and β-catenin in gastric cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0684-8

    DUXAP10 interacts PRC2/LSD1 and HuR to regulate KLF2, LATS1and β-catenin expression. a QPCR was used to examine the levels of potential DUXAP10 targets in GC cells after knockdown of DUXAP10. b KLF2, LATS1and β-catenin protein levels were analyzed by western blot in GC cells after knockdown of DUXAP10. c KLF2 and LATS1 levels were analyzed by qPCR in GC cells after transfection with EZH2, SUZ12 and LSD1 siRNA. d , e ChIP shows SUZ12, EZH2, H3K27me3, LSD1 and H3K4me2 occupancy in the LATS1 and KLF2 promoter region, while knockdown of DUXAP10 decreased their binding ability. f RIP assays show the interaction between HuR and β-catenin mRNA in GC cells, and knockdown of DUXAP10 impaired their interaction ability. * P
    Figure Legend Snippet: DUXAP10 interacts PRC2/LSD1 and HuR to regulate KLF2, LATS1and β-catenin expression. a QPCR was used to examine the levels of potential DUXAP10 targets in GC cells after knockdown of DUXAP10. b KLF2, LATS1and β-catenin protein levels were analyzed by western blot in GC cells after knockdown of DUXAP10. c KLF2 and LATS1 levels were analyzed by qPCR in GC cells after transfection with EZH2, SUZ12 and LSD1 siRNA. d , e ChIP shows SUZ12, EZH2, H3K27me3, LSD1 and H3K4me2 occupancy in the LATS1 and KLF2 promoter region, while knockdown of DUXAP10 decreased their binding ability. f RIP assays show the interaction between HuR and β-catenin mRNA in GC cells, and knockdown of DUXAP10 impaired their interaction ability. * P

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

    15) Product Images from "Aging Suppresses Skin-Derived Circulating SDF1 to Promote Full-Thickness Tissue Regeneration"

    Article Title: Aging Suppresses Skin-Derived Circulating SDF1 to Promote Full-Thickness Tissue Regeneration

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.08.054

    Mouse and Human Skin Exhibit Age-Dependent EZH2-Mediated SDF1 Induction (A) Age-dependent epigenetic regulation of SDF1. Shown are H3K27me3, H3K4me3, and EZH2 chromatin immune precipitation of ear wound edge tissue at baseline and 1 week post-injury at 3 different locations on the SDF1 gene. n = 6. *p
    Figure Legend Snippet: Mouse and Human Skin Exhibit Age-Dependent EZH2-Mediated SDF1 Induction (A) Age-dependent epigenetic regulation of SDF1. Shown are H3K27me3, H3K4me3, and EZH2 chromatin immune precipitation of ear wound edge tissue at baseline and 1 week post-injury at 3 different locations on the SDF1 gene. n = 6. *p

    Techniques Used:

    16) Product Images from "BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A"

    Article Title: BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr2354

    EZH2 is overexpressed in BRCA1-deficient human breast tumors. (a) The mean (± standard error of the mean) log10 ratio of EZH2 expression in human breast cancer samples [ 45 ] is -0.15 (± 0.041; > five-year survival, good prognosis), 0.028 (± 0.046;
    Figure Legend Snippet: EZH2 is overexpressed in BRCA1-deficient human breast tumors. (a) The mean (± standard error of the mean) log10 ratio of EZH2 expression in human breast cancer samples [ 45 ] is -0.15 (± 0.041; > five-year survival, good prognosis), 0.028 (± 0.046;

    Techniques Used: Expressing

    Restoration of BRCA1 partially rescues from sensitivity to DZNep. (a) Detection of the human BRCA1 allele by PCR amplification of exon 11 in the reconstituted subclones KB1PR-3.12 E3 and F4. The human breast cancer cell line T47D was used as a positive control. (b) EZH2 protein levels of (untreated) K K 14cre; B rca 1 F / F ; p 53 F / F (KB1P), KB1PR and K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP) cell lines were analysed by western blotting. Corresponding Ezh2 mRNA levels of KB1P, KB1PR and KP cell lines were measured by quantitative RT-PCR (shown as fold induction relative to Hprt ). (c) Representative growth inhibition curves for BRCA1-deficient cell lines (KB1P, in blue), BRCA1-proficient cell lines (KP, in red) and two clones of a BRCA1 -reconstituted cell line (KB1PR E3 and F4, in purple) treated with 3-deazaneplanocin A (DZNep). A serial dilution of DZNep was added to the cells and cell viability was measured five days later (mean ± standard error of the mean). (d) EZH2 protein levels of hBRCA1 -reconstituted KB1P cells 48 hours after treatment with DZNep or siRNAs targeting EZH2.
    Figure Legend Snippet: Restoration of BRCA1 partially rescues from sensitivity to DZNep. (a) Detection of the human BRCA1 allele by PCR amplification of exon 11 in the reconstituted subclones KB1PR-3.12 E3 and F4. The human breast cancer cell line T47D was used as a positive control. (b) EZH2 protein levels of (untreated) K K 14cre; B rca 1 F / F ; p 53 F / F (KB1P), KB1PR and K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP) cell lines were analysed by western blotting. Corresponding Ezh2 mRNA levels of KB1P, KB1PR and KP cell lines were measured by quantitative RT-PCR (shown as fold induction relative to Hprt ). (c) Representative growth inhibition curves for BRCA1-deficient cell lines (KB1P, in blue), BRCA1-proficient cell lines (KP, in red) and two clones of a BRCA1 -reconstituted cell line (KB1PR E3 and F4, in purple) treated with 3-deazaneplanocin A (DZNep). A serial dilution of DZNep was added to the cells and cell viability was measured five days later (mean ± standard error of the mean). (d) EZH2 protein levels of hBRCA1 -reconstituted KB1P cells 48 hours after treatment with DZNep or siRNAs targeting EZH2.

    Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control, Western Blot, Quantitative RT-PCR, Inhibition, Clone Assay, Serial Dilution

    Ezh2 expression is elevated in BRCA1-deficient primary mouse mammary tumors. (a) mRNA levels of Ezh2 in BRCA1-deficient ( K 14cre; B rca 1 F / F ; p 53 F / F (KB1P)) and BRCA1-proficient ( K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP)) mammary tumors analyzed by microarray analysis. The mean (± standard error of the mean) log2 ratio of Ezh2 expression in 21 KP tumors is -0.036 (± 0.067) and 0.497 (± 0.054) in 32 KB1P tumors. The Ezh2 expression is significantly higher in KB1P tumors compared with KP tumors (*Wilcoxon exact test). (b) EZH2 protein levels in two independent primary KB1P and in two independent primary KP tumors detected by immunohistochemistry (scale bar represents 100 μm), representative of a total of four tumors analysed for each genotype. (c) Quantification of EZH2 immunohistochemistry shown in b (* Wilcoxon exact test).
    Figure Legend Snippet: Ezh2 expression is elevated in BRCA1-deficient primary mouse mammary tumors. (a) mRNA levels of Ezh2 in BRCA1-deficient ( K 14cre; B rca 1 F / F ; p 53 F / F (KB1P)) and BRCA1-proficient ( K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP)) mammary tumors analyzed by microarray analysis. The mean (± standard error of the mean) log2 ratio of Ezh2 expression in 21 KP tumors is -0.036 (± 0.067) and 0.497 (± 0.054) in 32 KB1P tumors. The Ezh2 expression is significantly higher in KB1P tumors compared with KP tumors (*Wilcoxon exact test). (b) EZH2 protein levels in two independent primary KB1P and in two independent primary KP tumors detected by immunohistochemistry (scale bar represents 100 μm), representative of a total of four tumors analysed for each genotype. (c) Quantification of EZH2 immunohistochemistry shown in b (* Wilcoxon exact test).

    Techniques Used: Expressing, Microarray, Immunohistochemistry

    EZH2 is required for survival of BRCA1-deficient but not BRCA1-proficient tumors cells. (a) Ezh2 mRNA expression measured by quantitative RT-PCR in BRCA1-deficient cells (in red, K 14cre; B rca 1 F / F ; p 53 F / F (KB1P)) and in BRCA1-proficient cells (in blue, K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP)) after treatment with siRNAs against Ezh2 or 5 μM 3-deazaneplanocin A (DZNep; shown as fold induction relative to Hprt ). (b) EZH2 levels analysed by western blot after KB1P and KP cells were treated with siRNAs targeting Ezh2 or non-targeting siRNAs (siNTC) and 5 μM DZNep or vehicle (dimethyl sulfoxide (DMSO)) for the indicated period. (c) Phase-contrast images of BRCA1-deficient and BRCA1-proficient cells treated for 48 hours with control (ctrl) siRNAs, siRNAs against Ezh2 or 5 μM DZNep (original magnification 10×). (d) Growth curves of KB1P (depicted in red) and KP cell lines (depicted in blue) treated with control (ctrl) siRNAs, siRNAs against Ezh2 or 5 μM DZNep. Data measured by Cell Titer Blue and represented as the mean ± standard error of the mean (three independent experiments). The depicted cell lines are representative for all three KB1P and KP cell lines.
    Figure Legend Snippet: EZH2 is required for survival of BRCA1-deficient but not BRCA1-proficient tumors cells. (a) Ezh2 mRNA expression measured by quantitative RT-PCR in BRCA1-deficient cells (in red, K 14cre; B rca 1 F / F ; p 53 F / F (KB1P)) and in BRCA1-proficient cells (in blue, K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP)) after treatment with siRNAs against Ezh2 or 5 μM 3-deazaneplanocin A (DZNep; shown as fold induction relative to Hprt ). (b) EZH2 levels analysed by western blot after KB1P and KP cells were treated with siRNAs targeting Ezh2 or non-targeting siRNAs (siNTC) and 5 μM DZNep or vehicle (dimethyl sulfoxide (DMSO)) for the indicated period. (c) Phase-contrast images of BRCA1-deficient and BRCA1-proficient cells treated for 48 hours with control (ctrl) siRNAs, siRNAs against Ezh2 or 5 μM DZNep (original magnification 10×). (d) Growth curves of KB1P (depicted in red) and KP cell lines (depicted in blue) treated with control (ctrl) siRNAs, siRNAs against Ezh2 or 5 μM DZNep. Data measured by Cell Titer Blue and represented as the mean ± standard error of the mean (three independent experiments). The depicted cell lines are representative for all three KB1P and KP cell lines.

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

    Chemical EZH2-inhibitor DZNep selectively kills BRCA1-deficient tumor cells. (a) Representative growth inhibition curves for BRCA1-deficient cell lines ( K 14cre; B rca 1 F / F ; p 53 F / F (KB1P), in blue) and BRCA1-proficient cell lines ( K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP), in red) treated with 3-deazaneplanocin A (DZNep). A serial dilution of DZNep was added to the cells and cell viability was measured five days later (each data point represents the mean ± standard error of the mean (SEM) of three independent experiments). (b) Representative growth inhibition curves for BRCA1-deficient cell lines (KB1P, in blue) and BRCA1-proficient cell lines (KP, in red) treated with trichostatin A (TSA). A serial dilution of TSA was added to the cells and cell viability was measured five days later (mean ± SEM).
    Figure Legend Snippet: Chemical EZH2-inhibitor DZNep selectively kills BRCA1-deficient tumor cells. (a) Representative growth inhibition curves for BRCA1-deficient cell lines ( K 14cre; B rca 1 F / F ; p 53 F / F (KB1P), in blue) and BRCA1-proficient cell lines ( K 14cre;Brca1 w . t / w . t ; p 53 F / F (KP), in red) treated with 3-deazaneplanocin A (DZNep). A serial dilution of DZNep was added to the cells and cell viability was measured five days later (each data point represents the mean ± standard error of the mean (SEM) of three independent experiments). (b) Representative growth inhibition curves for BRCA1-deficient cell lines (KB1P, in blue) and BRCA1-proficient cell lines (KP, in red) treated with trichostatin A (TSA). A serial dilution of TSA was added to the cells and cell viability was measured five days later (mean ± SEM).

    Techniques Used: Inhibition, Serial Dilution

    17) Product Images from "Polycomb Repressive Complex 1 generates discrete compacted domains that change during differentiation"

    Article Title: Polycomb Repressive Complex 1 generates discrete compacted domains that change during differentiation

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.01.009

    Chromatin is organized into self-interacting domains of local interactions at Hox gene clusters in ESC and requires canonical PRC1 A , Schematic - Canonical and variant PRC1 complexes have distinct subunit composition but are found to co-exist at PRC1 targets. B , CRISPR-Cas9 targeting of Phc1 leads to loss of Phc1 protein expression. Phc2 expression is not increased to compensate for Phc1-loss. Ezh2 and Actin are controls. C , RNA-Seq shows that Hox cluster genes, which are PRC1 targets, change gene expression in Phc1-KO ESC. Orange – PRC1 occupied genes in WT ESC. Blue – Hox cluster genes. D , ChIP-PCR at select PRC1 target loci (GD – gene desert control) show loss of canonical PRC1 subunits Phc1 and Cbx7, approximately 50% reduction in Ring1b occupancy and no significant change in Kdm2b. Data represented as mean +/- SD. E-F .
    Figure Legend Snippet: Chromatin is organized into self-interacting domains of local interactions at Hox gene clusters in ESC and requires canonical PRC1 A , Schematic - Canonical and variant PRC1 complexes have distinct subunit composition but are found to co-exist at PRC1 targets. B , CRISPR-Cas9 targeting of Phc1 leads to loss of Phc1 protein expression. Phc2 expression is not increased to compensate for Phc1-loss. Ezh2 and Actin are controls. C , RNA-Seq shows that Hox cluster genes, which are PRC1 targets, change gene expression in Phc1-KO ESC. Orange – PRC1 occupied genes in WT ESC. Blue – Hox cluster genes. D , ChIP-PCR at select PRC1 target loci (GD – gene desert control) show loss of canonical PRC1 subunits Phc1 and Cbx7, approximately 50% reduction in Ring1b occupancy and no significant change in Kdm2b. Data represented as mean +/- SD. E-F .

    Techniques Used: Variant Assay, CRISPR, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    18) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    19) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    20) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    21) Product Images from "Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1"

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501466

    EZH2 directly silences IRF8 expression
    Figure Legend Snippet: EZH2 directly silences IRF8 expression

    Techniques Used: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression
    Figure Legend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Techniques Used: Expressing

    22) Product Images from "Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1"

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501466

    EZH2 directly silences IRF8 expression
    Figure Legend Snippet: EZH2 directly silences IRF8 expression

    Techniques Used: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression
    Figure Legend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Techniques Used: Expressing

    23) Product Images from "Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1"

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501466

    EZH2 directly silences IRF8 expression
    Figure Legend Snippet: EZH2 directly silences IRF8 expression

    Techniques Used: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression
    Figure Legend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Techniques Used: Expressing

    24) Product Images from "Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming"

    Article Title: Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming

    Journal: Scientific Reports

    doi: 10.1038/srep08229

    Inactivation of Ezh2 or inhibition of H3K27me3 activity enhances TGF-β signaling in human fibroblasts. (a) Expression profile of TGF-βR2 and CDH1 in hFibs undergoing reprogramming at various time courses indicated. (b) Expression profile of mRNAs encoding TGF-β receptors in hFibs transduced with control and Ezh2 shRNA. (c) Western blotting for TGF-βR2 and GAPDH control in hFibs transduced with control and Ezh2 shRNA. Uncropped/Full blots images are provided in Supplementary Fig. 7 a–b . (d) Expression profile of mRNAs encoding TGF-β receptors in DMSO and GSK treated human fibroblasts. (e) Western blotting using anti phospho- Smad2 and total Smad2 antibody in hFibs transduced with control and Ezh2 shRNA. Uncropped/Full blots images are provided in Supplementary Fig. 7 c–d (f) Relative expression of mRNA for Twist, Snail and E-cadherin (CDH1) that are downstream targets for TGF-β signaling in hFibs transduced with control and Ezh2 shRNA.
    Figure Legend Snippet: Inactivation of Ezh2 or inhibition of H3K27me3 activity enhances TGF-β signaling in human fibroblasts. (a) Expression profile of TGF-βR2 and CDH1 in hFibs undergoing reprogramming at various time courses indicated. (b) Expression profile of mRNAs encoding TGF-β receptors in hFibs transduced with control and Ezh2 shRNA. (c) Western blotting for TGF-βR2 and GAPDH control in hFibs transduced with control and Ezh2 shRNA. Uncropped/Full blots images are provided in Supplementary Fig. 7 a–b . (d) Expression profile of mRNAs encoding TGF-β receptors in DMSO and GSK treated human fibroblasts. (e) Western blotting using anti phospho- Smad2 and total Smad2 antibody in hFibs transduced with control and Ezh2 shRNA. Uncropped/Full blots images are provided in Supplementary Fig. 7 c–d (f) Relative expression of mRNA for Twist, Snail and E-cadherin (CDH1) that are downstream targets for TGF-β signaling in hFibs transduced with control and Ezh2 shRNA.

    Techniques Used: Inhibition, Activity Assay, Expressing, Transduction, shRNA, Western Blot

    Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation. (a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c . (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f . (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.
    Figure Legend Snippet: Inhibition of H3K27me3 activity impairs mesenchymal to epithelial transition during iPSC generation. (a) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in hFibs treated with H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 a-c . (b) Relative expression of p53 and p21 mRNA in DMSO control and GSK treated hFibs. (c) Representative bright field and β- gal staining images upon treatment of hFibs with DMSO and GSK-126. (d) Western blot analysis using anti Ezh2, anti-H3K27Me3 and control anti-pan H3 antibodies in H9 hESC treated with DMSO or H3K27me3 inhibitor GSK-126. Uncropped/Full blots images are provided in Supplementary Fig. 6 d–f . (e) Tra 1-60 DAB staining in IMR-90 iPSC, H9 hESC cultures treated with DMSO and GSK-126. (f) Schematic representation of reprogramming protocol and time points of addition of GSK to the reprogramming cultures. (g) Total number of reprogrammed colonies generated three weeks post OSKM transduced hFibs and upon GSK treatment at indicated time points in schema (h) Tra 1-60 DAB staining on day 21 post addition of reprogramming factors and GSK treatment (i) E-cad DAB staining on day 10 post addition of reprogramming factors and GSK treatment as indicated in schema.

    Techniques Used: Inhibition, Activity Assay, Western Blot, Expressing, Staining, Generated

    Ezh2 interacts with c-Myc and Sox2 to repress TGF-β receptor and p53 activity during reprogramming. (a) Cell extracts from hFibs transduced with OSKM (day 7) were subjected to immunoprecipitation using Ezh2 or control IgG followed by western blotting for Oct4, Sox2 and c-Myc. Uncropped/Full blots images are provided in Supplementary Fig. 8 a–c (b) Cell extracts from H9-hESC were subjected to immunoprecipitation using Ezh1, Ezh2 or control antibodies followed by immunoblotting for indicated antibodies. Uncropped/Full blots images are provided in Supplementary Fig. 8 d–f . (c–d) Chromatin immunoprecipitation for Sox2, c-Myc and Ezh2 were performed from hFibs, OSKM transduced hFibs at day 4 and 7 days followed by quantitative PCR amplification of indicated gene promoters. (e) Chromatin IP for H3K27me3 was performed in hFibs, OSKM transduced hFibs at day 7 and iPSC.
    Figure Legend Snippet: Ezh2 interacts with c-Myc and Sox2 to repress TGF-β receptor and p53 activity during reprogramming. (a) Cell extracts from hFibs transduced with OSKM (day 7) were subjected to immunoprecipitation using Ezh2 or control IgG followed by western blotting for Oct4, Sox2 and c-Myc. Uncropped/Full blots images are provided in Supplementary Fig. 8 a–c (b) Cell extracts from H9-hESC were subjected to immunoprecipitation using Ezh1, Ezh2 or control antibodies followed by immunoblotting for indicated antibodies. Uncropped/Full blots images are provided in Supplementary Fig. 8 d–f . (c–d) Chromatin immunoprecipitation for Sox2, c-Myc and Ezh2 were performed from hFibs, OSKM transduced hFibs at day 4 and 7 days followed by quantitative PCR amplification of indicated gene promoters. (e) Chromatin IP for H3K27me3 was performed in hFibs, OSKM transduced hFibs at day 7 and iPSC.

    Techniques Used: Activity Assay, Transduction, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

    Ezh2 regulates miR-27a expression during human fibroblasts reprogramming. (a) Differentially expressed miRNAs in sh-Ezh2 hFibs calculated by Edge R and DESeq programmes. (b) Histogram depicting the number of miRNA regulating indicated pathways in shEzh2 hFibs. (c) qPCR results of indicated miRNA expression levels in shCnt, shEzh2 and Ezh2 OE hFibs. (d) qPCR results of indicated miRNA expression levels in DMSO control and GSK treated hFibs. (e) Expression profile of indicated miRNA in hFibs, OSKM transduced hFibs on day7 and iPSCs. (f) Chromatin IP for Ezh2 and H3K27me3 was performed in hFbs, OSKM transduced hFibs at day7 and iPSC. Two Kilobase upstream sequence of miR27a was amplified from immunoprecipitated material. (g) Schematic representation of miR-27 plasmid overexpression and OSKM transduction. E-cadherin DAB staining was performed 10 days post introduction of reprogramming factors. (h) E-cad DAB staining in H9 hESC, control and miR-27a overexpressing hFibs.
    Figure Legend Snippet: Ezh2 regulates miR-27a expression during human fibroblasts reprogramming. (a) Differentially expressed miRNAs in sh-Ezh2 hFibs calculated by Edge R and DESeq programmes. (b) Histogram depicting the number of miRNA regulating indicated pathways in shEzh2 hFibs. (c) qPCR results of indicated miRNA expression levels in shCnt, shEzh2 and Ezh2 OE hFibs. (d) qPCR results of indicated miRNA expression levels in DMSO control and GSK treated hFibs. (e) Expression profile of indicated miRNA in hFibs, OSKM transduced hFibs on day7 and iPSCs. (f) Chromatin IP for Ezh2 and H3K27me3 was performed in hFbs, OSKM transduced hFibs at day7 and iPSC. Two Kilobase upstream sequence of miR27a was amplified from immunoprecipitated material. (g) Schematic representation of miR-27 plasmid overexpression and OSKM transduction. E-cadherin DAB staining was performed 10 days post introduction of reprogramming factors. (h) E-cad DAB staining in H9 hESC, control and miR-27a overexpressing hFibs.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Sequencing, Amplification, Immunoprecipitation, Plasmid Preparation, Over Expression, Transduction, Staining

    Proposed model by which Ezh2 regulates initiation events in favor of fibroblast transition towards pluripotent state.
    Figure Legend Snippet: Proposed model by which Ezh2 regulates initiation events in favor of fibroblast transition towards pluripotent state.

    Techniques Used:

    25) Product Images from "The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state"

    Article Title: The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt285

    shRNA-mediated knockdown of Safb1 does not alter MyoD recruitment to myogenic sequences but increases Polycomb repressive marks. Proliferating (GM) or 72 h differentiated (DM) C2C12 cells expressing control shRNA or shRNA against Safb1 were fixed and processed for ChIP assays using ( A ) antibodies against MyoD and Safb1 (top panel) or ( B ) antibodies against Ezh2 and H3K27me3 at the indicated gene regulatory sequences. H3K27me3 enrichment is presented on the right-hand y-axis in the bottom panel. Data represent the mean of three independent experiments ± SEM. t test analyses indicate that when comparing binding in sh-Safb1-treated differentiated cells with sh-Control differentiated cells, the binding of Safb1 was significantly reduced ( P
    Figure Legend Snippet: shRNA-mediated knockdown of Safb1 does not alter MyoD recruitment to myogenic sequences but increases Polycomb repressive marks. Proliferating (GM) or 72 h differentiated (DM) C2C12 cells expressing control shRNA or shRNA against Safb1 were fixed and processed for ChIP assays using ( A ) antibodies against MyoD and Safb1 (top panel) or ( B ) antibodies against Ezh2 and H3K27me3 at the indicated gene regulatory sequences. H3K27me3 enrichment is presented on the right-hand y-axis in the bottom panel. Data represent the mean of three independent experiments ± SEM. t test analyses indicate that when comparing binding in sh-Safb1-treated differentiated cells with sh-Control differentiated cells, the binding of Safb1 was significantly reduced ( P

    Techniques Used: shRNA, Expressing, Chromatin Immunoprecipitation, Binding Assay

    26) Product Images from "Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma"

    Article Title: Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171031

    ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P
    Figure Legend Snippet: ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P

    Techniques Used: Expressing, Western Blot, In Vitro, Labeling, Negative Control, Quantitative RT-PCR, Positive Control, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    27) Product Images from "The long non‐coding RNA PVT1 represses ANGPTL4 transcription through binding with EZH2 in trophoblast cell"

    Article Title: The long non‐coding RNA PVT1 represses ANGPTL4 transcription through binding with EZH2 in trophoblast cell

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13405

    PVT 1 binds to Ezh2 to suppress ANGPTL 4 expression. ( A ) Using qPCR , relative PVT 1 levels are mostly located in nucleus, in which GAPDH and U1 acted as the marker of cytoplasm and nucleus, respectively. ( B ) RIP assays established that PVT 1 could interact with Ezh2. ( C ) Knockdown EZH 2 triggered ANGPTL 4 expression at the mRNA levels by qPCR and protein levels by WB . ( D ) Ch IP assays uncovered that Ezh2 and H3K27me3 were enriched in the promoter region of ANGPTL 4 , and this enrichment was reduced after PVT 1 knockdown. * P
    Figure Legend Snippet: PVT 1 binds to Ezh2 to suppress ANGPTL 4 expression. ( A ) Using qPCR , relative PVT 1 levels are mostly located in nucleus, in which GAPDH and U1 acted as the marker of cytoplasm and nucleus, respectively. ( B ) RIP assays established that PVT 1 could interact with Ezh2. ( C ) Knockdown EZH 2 triggered ANGPTL 4 expression at the mRNA levels by qPCR and protein levels by WB . ( D ) Ch IP assays uncovered that Ezh2 and H3K27me3 were enriched in the promoter region of ANGPTL 4 , and this enrichment was reduced after PVT 1 knockdown. * P

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

    28) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    29) Product Images from "Long non-coding RNA LUCAT1 is associated with poor prognosis in human non-small cell lung cancer and regulates cell proliferation via epigenetically repressing p21 and p57 expression"

    Article Title: Long non-coding RNA LUCAT1 is associated with poor prognosis in human non-small cell lung cancer and regulates cell proliferation via epigenetically repressing p21 and p57 expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16044

    LUCAT1 is required to target EZH2 occupancy and activity to epigenetically regulate the expression of CKIs ( A ) After si-LUCAT1 3# transfection, the expression of p15, p16, p21, p27, and p57 were determined by qPCR and western blot assays in A549 and SPC-A1 cells. ( B ) After si-EZH2 or si-SUZ12 transfection, the expression of p21 and p57 were detected by qPCR in A549 and SPC-A1 cells. And western blot assay was performed to detect the protein level of p21 and p57 after si-EZH2 transfection. ( C ) ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the p21 and p57 locus after siRNA treatment targeting si-NC or si-LUCAT1 3# in SPC-A1 cells. Antibody enrichment was quantified relative to input controls. Antibody directed against IgG was used as a negative control. Error bars indicate means ± S.E.M. * P
    Figure Legend Snippet: LUCAT1 is required to target EZH2 occupancy and activity to epigenetically regulate the expression of CKIs ( A ) After si-LUCAT1 3# transfection, the expression of p15, p16, p21, p27, and p57 were determined by qPCR and western blot assays in A549 and SPC-A1 cells. ( B ) After si-EZH2 or si-SUZ12 transfection, the expression of p21 and p57 were detected by qPCR in A549 and SPC-A1 cells. And western blot assay was performed to detect the protein level of p21 and p57 after si-EZH2 transfection. ( C ) ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the p21 and p57 locus after siRNA treatment targeting si-NC or si-LUCAT1 3# in SPC-A1 cells. Antibody enrichment was quantified relative to input controls. Antibody directed against IgG was used as a negative control. Error bars indicate means ± S.E.M. * P

    Techniques Used: Activity Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Negative Control

    Subcellular fractionation location of LUCAT1, and LUCAT1 could bind to PRC2 ( A ) LUCAT1 nuclear localization, as identified using qRT-PCR in fractionated A549 and SPC-A1 cells. After nuclear and cytosolic separation, RNA expression was measured by qRT-PCR. GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. ( B ) RIP experiments were performed in A549 and SPC-A1 cells and the coprecipitated RNA were subjected to qRT-PCR for LUCAT1. The fold enrichment of LUCAT1 in EZH2/SUZ12 RIP is relative to its matching IgG control RIP. ( C ) The level of LUCAT1 expression in NSCLC tissues showed a statistically positive correlation with the relative level of EZH2 and SUZ12 expression ( N = 68). * P
    Figure Legend Snippet: Subcellular fractionation location of LUCAT1, and LUCAT1 could bind to PRC2 ( A ) LUCAT1 nuclear localization, as identified using qRT-PCR in fractionated A549 and SPC-A1 cells. After nuclear and cytosolic separation, RNA expression was measured by qRT-PCR. GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. ( B ) RIP experiments were performed in A549 and SPC-A1 cells and the coprecipitated RNA were subjected to qRT-PCR for LUCAT1. The fold enrichment of LUCAT1 in EZH2/SUZ12 RIP is relative to its matching IgG control RIP. ( C ) The level of LUCAT1 expression in NSCLC tissues showed a statistically positive correlation with the relative level of EZH2 and SUZ12 expression ( N = 68). * P

    Techniques Used: Fractionation, Quantitative RT-PCR, RNA Expression, Marker, Expressing

    30) Product Images from "Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition"

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition

    Journal: Nature medicine

    doi: 10.1038/nm.4415

    Epigenetic GSC signatures in multi-regional primary specimens ( a ) Immunofluorescence images showing CD31 (red), CA9 (red), CD15 (red), H3K27Me3 (green), and H2AK119Ub (green) positive location and cells in multiregional glioblastoma samples. Scale bar, 25 μ m. ( b ) Overlap in regional specific H3K27me3 or H2AK119Ub binding genes in CW2451- and CW2473-derived CD15 positive glioma cells. Venn diagrams showing overlaps between region specific peaks derived from H3K27me3 or H2AK119Ub ChIP-seq experiments on two primary glioblastoma specimens. ( c ) Pie graph showing fraction of regional unique target genes by H3K27Me3 or H2AK119Ub from the overlapping region specific genes shown in ( b ). ( d ) Gene ontology bubble plots showing gene signatures enriched in the overlapping region specific peaks shown in ( b ). ( e, f ) Enrichment levels of EZH2 ( e ) or BMI1 ( f ) activation signatures in transcriptional subgroups from the TCGA glioblastoma dataset (n = 10, Non-tumor; n = 161, Proneural; n = 209, Classical; n = 167, Mesenchymal). Data shows the median value (black bars). **, p
    Figure Legend Snippet: Epigenetic GSC signatures in multi-regional primary specimens ( a ) Immunofluorescence images showing CD31 (red), CA9 (red), CD15 (red), H3K27Me3 (green), and H2AK119Ub (green) positive location and cells in multiregional glioblastoma samples. Scale bar, 25 μ m. ( b ) Overlap in regional specific H3K27me3 or H2AK119Ub binding genes in CW2451- and CW2473-derived CD15 positive glioma cells. Venn diagrams showing overlaps between region specific peaks derived from H3K27me3 or H2AK119Ub ChIP-seq experiments on two primary glioblastoma specimens. ( c ) Pie graph showing fraction of regional unique target genes by H3K27Me3 or H2AK119Ub from the overlapping region specific genes shown in ( b ). ( d ) Gene ontology bubble plots showing gene signatures enriched in the overlapping region specific peaks shown in ( b ). ( e, f ) Enrichment levels of EZH2 ( e ) or BMI1 ( f ) activation signatures in transcriptional subgroups from the TCGA glioblastoma dataset (n = 10, Non-tumor; n = 161, Proneural; n = 209, Classical; n = 167, Mesenchymal). Data shows the median value (black bars). **, p

    Techniques Used: Immunofluorescence, Binding Assay, Derivative Assay, Chromatin Immunoprecipitation, Activation Assay

    In vivo therapeutic efficacy of combined pharmacologic inhibition of BMI1 and EZH2 on subtype-mixed glioblastoma model ( a ) Experimental design for in vivo effects of BMI1 (PTC596) and EZH2 (EPZ6438) inhibitors on xenograft of mixed proneural and mesenchymal GSCs. ( b ) Bioluminescence images of mice bearing mixed proneural and mesenchymal xenografts derived from luciferase-expressing PN1919 and MES20 cells, showing the effect of combined treatment of 10 mg/kg BMI1-i per week and 350 mg/kg EZH2-i thrice weekly on tumor growth. Right panels: Quantification of bioluminescence signals during 45 days of treatment in mice implanted with luciferase-expressing PN1919 and MES20 cells. The signals were normalized to day 10 signaling intensity for each mouse ( n = 10 per group and time point). *, p
    Figure Legend Snippet: In vivo therapeutic efficacy of combined pharmacologic inhibition of BMI1 and EZH2 on subtype-mixed glioblastoma model ( a ) Experimental design for in vivo effects of BMI1 (PTC596) and EZH2 (EPZ6438) inhibitors on xenograft of mixed proneural and mesenchymal GSCs. ( b ) Bioluminescence images of mice bearing mixed proneural and mesenchymal xenografts derived from luciferase-expressing PN1919 and MES20 cells, showing the effect of combined treatment of 10 mg/kg BMI1-i per week and 350 mg/kg EZH2-i thrice weekly on tumor growth. Right panels: Quantification of bioluminescence signals during 45 days of treatment in mice implanted with luciferase-expressing PN1919 and MES20 cells. The signals were normalized to day 10 signaling intensity for each mouse ( n = 10 per group and time point). *, p

    Techniques Used: In Vivo, Inhibition, Mouse Assay, Derivative Assay, Luciferase, Expressing

    Differential polycomb repressive complex function in glioblastoma subgroups ( a ) Representative images of glioblastoma tissue microarray samples (n = 96) showing the expression of EZH2, BMI1, CD44, and OLIG2. Scale bar, 500 μ m. ( b ) Kaplan-Meier curve of patient survival stratified by EZH2 and BMI1 protein expression level from a glioblastoma tissue microarray. Log-rank p-values were used to determine statistics significance. ( c ) Cell lysates were prepared from neural progenitor cells (NPC1, NPC2, and NPC3), proneural GSCs 670 (PN11, PN23, PN1919, and PN3691), and mesenchymal GSCs (MES20, MES3565, MES28, MES738). Protein was resolved by SDS-PAGE. Immunoblot for performed for BMI1, H2K119Ub, EZH2, H3K27me3, and markers for mesenchymal (CD44 and YKL40) and proneural (OLIG2 and SOX2) glioblastoma. ( d ) Immunoprecipitation followed by immunoblot was performed for BMI1 polyubiquitination in PN1919 and MES28 cells in presence or absence of lactacystin treatment for 5 hours (Lacta, 10 μ M). Right panel: quantification of BMI1 polyubiquitination by Image J. Data normalized by input loading controls. ( e ) Rank-ordered list of correlation coefficients ( r values) between ubiquitin ligases and BMI1 activation or inhibition signatures in TCGA glioblastoma samples. ( f ) BMI1 polyubiquitination by RNF144A in PN1919 cells after transduction with shRNA control (shCTRL) or shRNF144A (shRNF144A-1099 and shRNF144A-3112) was examined by immunoprecipitation of BMI1 followed by immunoblotting for BMI1 or ubiquitin in presence or absence of lactacystin treatment for 5 hours (Lacta, 10 μ M). Levels of BMI1, RNF144A, and TUBULIN were measured by immunoblot in the input whole cell lysates. ( g ) Neural progenitor cells (NPC1), proneural GSCs (PN1919 and PN3691), and mesenchymal GSCs (MES20 and MES28) were grown under baseline, low-glucose, hypoxia, or combined conditions. Levels of RNF144A, BMI1, EZH2, CD44, YKL40, OLIG2, and SOX2 proteins were measured by immunoblot. ( h ) Cell viability of neural progenitor cells (NPC1), proneural GSCs (PN11, PN23, PN1919, and PN3691), and mesenchymal GSCs (MES20, MES28, MES3565, and MES738) were determined under baseline, low-glucose, hypoxia, or combined conditions. Data are presented as mean ± SEM. *, p
    Figure Legend Snippet: Differential polycomb repressive complex function in glioblastoma subgroups ( a ) Representative images of glioblastoma tissue microarray samples (n = 96) showing the expression of EZH2, BMI1, CD44, and OLIG2. Scale bar, 500 μ m. ( b ) Kaplan-Meier curve of patient survival stratified by EZH2 and BMI1 protein expression level from a glioblastoma tissue microarray. Log-rank p-values were used to determine statistics significance. ( c ) Cell lysates were prepared from neural progenitor cells (NPC1, NPC2, and NPC3), proneural GSCs 670 (PN11, PN23, PN1919, and PN3691), and mesenchymal GSCs (MES20, MES3565, MES28, MES738). Protein was resolved by SDS-PAGE. Immunoblot for performed for BMI1, H2K119Ub, EZH2, H3K27me3, and markers for mesenchymal (CD44 and YKL40) and proneural (OLIG2 and SOX2) glioblastoma. ( d ) Immunoprecipitation followed by immunoblot was performed for BMI1 polyubiquitination in PN1919 and MES28 cells in presence or absence of lactacystin treatment for 5 hours (Lacta, 10 μ M). Right panel: quantification of BMI1 polyubiquitination by Image J. Data normalized by input loading controls. ( e ) Rank-ordered list of correlation coefficients ( r values) between ubiquitin ligases and BMI1 activation or inhibition signatures in TCGA glioblastoma samples. ( f ) BMI1 polyubiquitination by RNF144A in PN1919 cells after transduction with shRNA control (shCTRL) or shRNF144A (shRNF144A-1099 and shRNF144A-3112) was examined by immunoprecipitation of BMI1 followed by immunoblotting for BMI1 or ubiquitin in presence or absence of lactacystin treatment for 5 hours (Lacta, 10 μ M). Levels of BMI1, RNF144A, and TUBULIN were measured by immunoblot in the input whole cell lysates. ( g ) Neural progenitor cells (NPC1), proneural GSCs (PN1919 and PN3691), and mesenchymal GSCs (MES20 and MES28) were grown under baseline, low-glucose, hypoxia, or combined conditions. Levels of RNF144A, BMI1, EZH2, CD44, YKL40, OLIG2, and SOX2 proteins were measured by immunoblot. ( h ) Cell viability of neural progenitor cells (NPC1), proneural GSCs (PN11, PN23, PN1919, and PN3691), and mesenchymal GSCs (MES20, MES28, MES3565, and MES738) were determined under baseline, low-glucose, hypoxia, or combined conditions. Data are presented as mean ± SEM. *, p

    Techniques Used: Microarray, Expressing, SDS Page, Immunoprecipitation, Activation Assay, Inhibition, Transduction, shRNA

    Differential efficacy of BMI1 and EZH2 inhibitors against glioblastoma subgroups ( a ) Cell growth rates of GSCs transduced with shRNA control (shCNTRL), EZH2-knockdown (shEZH2-950 and shEZH2-2450) or BMI1-knockdown (shBMI1-880 and shBMI1-939) ( n = 5 per group and time point). Data shows mean ± SEM. **, p
    Figure Legend Snippet: Differential efficacy of BMI1 and EZH2 inhibitors against glioblastoma subgroups ( a ) Cell growth rates of GSCs transduced with shRNA control (shCNTRL), EZH2-knockdown (shEZH2-950 and shEZH2-2450) or BMI1-knockdown (shBMI1-880 and shBMI1-939) ( n = 5 per group and time point). Data shows mean ± SEM. **, p

    Techniques Used: Transduction, shRNA

    31) Product Images from "Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma"

    Article Title: Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171031

    ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P
    Figure Legend Snippet: ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P

    Techniques Used: Expressing, Western Blot, In Vitro, Labeling, Negative Control, Quantitative RT-PCR, Positive Control, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    32) 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

    33) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    34) Product Images from "Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1"

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501466

    EZH2 directly silences IRF8 expression
    Figure Legend Snippet: EZH2 directly silences IRF8 expression

    Techniques Used: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression
    Figure Legend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Techniques Used: Expressing

    35) Product Images from "Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1"

    Article Title: Cutting Edge: EZH2 promotes osteoclastogenesis by epigenetic silencing of the negative regulator IRF8 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501466

    EZH2 directly silences IRF8 expression
    Figure Legend Snippet: EZH2 directly silences IRF8 expression

    Techniques Used: Expressing

    EZH2 is required for osteoclast differentiation and NFATc1 expression
    Figure Legend Snippet: EZH2 is required for osteoclast differentiation and NFATc1 expression

    Techniques Used: Expressing

    36) Product Images from "Non-coding RNAs and LRRFIP1 Regulate TNF Expression"

    Article Title: Non-coding RNAs and LRRFIP1 Regulate TNF Expression

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1302063

    Knockdown of SUZ12 and EZH2 impairs LRRFIP1 binding
    Figure Legend Snippet: Knockdown of SUZ12 and EZH2 impairs LRRFIP1 binding

    Techniques Used: Binding Assay

    37) Product Images from "Non-coding RNAs and LRRFIP1 Regulate TNF Expression 1"

    Article Title: Non-coding RNAs and LRRFIP1 Regulate TNF Expression 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1302063

    Knockdown of SUZ12 and EZH2 impairs LRRFIP1 binding
    Figure Legend Snippet: Knockdown of SUZ12 and EZH2 impairs LRRFIP1 binding

    Techniques Used: Binding Assay

    38) Product Images from "Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma"

    Article Title: Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171031

    ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P
    Figure Legend Snippet: ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P

    Techniques Used: Expressing, Western Blot, In Vitro, Labeling, Negative Control, Quantitative RT-PCR, Positive Control, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    39) Product Images from "Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma"

    Article Title: Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171031

    ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P
    Figure Legend Snippet: ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P

    Techniques Used: Expressing, Western Blot, In Vitro, Labeling, Negative Control, Quantitative RT-PCR, Positive Control, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    40) Product Images from "Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma"

    Article Title: Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171031

    ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P
    Figure Legend Snippet: ILF3-AS1 negatively regulates miR-200b/a/429 expression via interacting with EZH2 ( A ) RNA pull-down assays followed by Western blot analysis revealed the specific enrichment of EZH2, but not GAPDH protein with in vitro transcribed biotin-labeled ILF3-AS1 compared with antisense RNA (negative control). ( B ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1, but not GAPDH mRNA with EZH2 antibody compared with nonspecific IgG (negative control). HEIH was used as positive control. ( C ) The specific binding of EZH2 and H3K27me3 levels across the miR-200b/a/429 promoter and the β-actin promoter in ILF3-AS1 stably depleted and control A375 cells were measured by ChIP assays followed by qPCR. ( D ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control A375 cells was measured by qRT-PCR. ( E ) The expression of miR-200b, miR-200a, and miR-429 in ILF3-AS1 stably depleted and control SK-MEL-2 cells was measured by qRT-PCR. Data are represented as mean ± SD; * P

    Techniques Used: Expressing, Western Blot, In Vitro, Labeling, Negative Control, Quantitative RT-PCR, Positive Control, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Related Articles

    Fluorescence:

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition
    Article Snippet: .. Immunofluorescence and β-GAL staining 10 μm thick slides of de-identified frozen multi-regional samples [glioblastoma patient (CW1757) according T1-weighted MRI images] and PN3691 (VEC/BMI1) xenografted frozen brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the primary antibodies against SOX2 (1:500, R & D system, AF2018), CD44 (1:100, BD Biosciences, 550392), vWF (1:500, Millipore, AB7356), CA9 (1:100, Genetex, GTX70020), H3K27Me3 (1:1000, Millipore, 07-689), H2AK119Ub (1:500, Cell signaling, #8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active CASPASE3 (1:100, Cell signaling, #9661S) and the secondary fluorescence-labeled antibodies. ..

    Immunolabeling:

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition
    Article Snippet: .. Immunofluorescence and β-GAL staining 10 μm thick slides of de-identified frozen multi-regional samples [glioblastoma patient (CW1757) according T1-weighted MRI images] and PN3691 (VEC/BMI1) xenografted frozen brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the primary antibodies against SOX2 (1:500, R & D system, AF2018), CD44 (1:100, BD Biosciences, 550392), vWF (1:500, Millipore, AB7356), CA9 (1:100, Genetex, GTX70020), H3K27Me3 (1:1000, Millipore, 07-689), H2AK119Ub (1:500, Cell signaling, #8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active CASPASE3 (1:100, Cell signaling, #9661S) and the secondary fluorescence-labeled antibodies. ..

    Blocking Assay:

    Article Title: Placental H3K27me3 establishes female resilience to prenatal insults
    Article Snippet: .. After running and transfer of proteins to a nitrocellulose membrane (Life Technologies), membranes were blocked with Odyssey blocking buffer (Li-Cor) and probed with rabbit anti-H3K27me3 (1:1000; Millipore 07–449), rabbit anti-H3K4me3 (1:1000, abcam ab8580), rabbit anti-H3K9ac (1:1000, Cell Signaling C5B11), rabbit anti-OGT (1:1000, Sigma DM-17), mouse anti-O-glycNac (1:1000, Biolegend CTD110.6), rabbit anti-EZH2 (1:2500; Millipore 07–689), and/or mouse anti-beta actin (1:30,000; Sigma A5441) followed by incubation in IRDye800-conjugated donkey anti-rabbit secondary (1:20,000; Li-Cor) and or IRDye680-conjugated goat anti-mouse secondary (1:20,000; Li-Cor). .. We used single-tailed t -tests for most two-group analyses because we had a priori hypotheses about the directionality of the changes in protein expression based on our previous data on sex differences and OGT regulation of H3K27me3 and predicted directional effects of Ezh2 and Ogt transgenic manipulation.

    Immunofluorescence:

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition
    Article Snippet: .. Immunofluorescence and β-GAL staining 10 μm thick slides of de-identified frozen multi-regional samples [glioblastoma patient (CW1757) according T1-weighted MRI images] and PN3691 (VEC/BMI1) xenografted frozen brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the primary antibodies against SOX2 (1:500, R & D system, AF2018), CD44 (1:100, BD Biosciences, 550392), vWF (1:500, Millipore, AB7356), CA9 (1:100, Genetex, GTX70020), H3K27Me3 (1:1000, Millipore, 07-689), H2AK119Ub (1:500, Cell signaling, #8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active CASPASE3 (1:100, Cell signaling, #9661S) and the secondary fluorescence-labeled antibodies. ..

    Immunoprecipitation:

    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
    Article Snippet: .. Antibodies The following antibodies were used for Chromatin Immunoprecipitation (ChIP), RNA-binding protein Immunoprecipitation (RIP) or immunoblotting: anti-H3K27me3 (17–622, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-EZH2 (07–689, Millipore), anti-SUZ12 (3737, Cell Signaling Technology), anti-EED (17–663, Millipore), anti-GAPDH (M20006, Abmart). .. Immunoblotting Cells were lysed for 1 h at 4°C in ice-cold lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 150 mM NaCl, 0.5 mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% protease inhibitor cocktail [Sigma], 1 mM sodium orthovanadate, 1 mM NaF, 1% [vol/vol] Triton X-100 and 10% [vol/vol] glycerol).

    Incubation:

    Article Title: Placental H3K27me3 establishes female resilience to prenatal insults
    Article Snippet: .. After running and transfer of proteins to a nitrocellulose membrane (Life Technologies), membranes were blocked with Odyssey blocking buffer (Li-Cor) and probed with rabbit anti-H3K27me3 (1:1000; Millipore 07–449), rabbit anti-H3K4me3 (1:1000, abcam ab8580), rabbit anti-H3K9ac (1:1000, Cell Signaling C5B11), rabbit anti-OGT (1:1000, Sigma DM-17), mouse anti-O-glycNac (1:1000, Biolegend CTD110.6), rabbit anti-EZH2 (1:2500; Millipore 07–689), and/or mouse anti-beta actin (1:30,000; Sigma A5441) followed by incubation in IRDye800-conjugated donkey anti-rabbit secondary (1:20,000; Li-Cor) and or IRDye680-conjugated goat anti-mouse secondary (1:20,000; Li-Cor). .. We used single-tailed t -tests for most two-group analyses because we had a priori hypotheses about the directionality of the changes in protein expression based on our previous data on sex differences and OGT regulation of H3K27me3 and predicted directional effects of Ezh2 and Ogt transgenic manipulation.

    Staining:

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition
    Article Snippet: .. Immunofluorescence and β-GAL staining 10 μm thick slides of de-identified frozen multi-regional samples [glioblastoma patient (CW1757) according T1-weighted MRI images] and PN3691 (VEC/BMI1) xenografted frozen brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the primary antibodies against SOX2 (1:500, R & D system, AF2018), CD44 (1:100, BD Biosciences, 550392), vWF (1:500, Millipore, AB7356), CA9 (1:100, Genetex, GTX70020), H3K27Me3 (1:1000, Millipore, 07-689), H2AK119Ub (1:500, Cell signaling, #8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active CASPASE3 (1:100, Cell signaling, #9661S) and the secondary fluorescence-labeled antibodies. ..

    Magnetic Resonance Imaging:

    Article Title: Targeting Glioma Stem Cells through Combined BMI1 and EZH2 Inhibition
    Article Snippet: .. Immunofluorescence and β-GAL staining 10 μm thick slides of de-identified frozen multi-regional samples [glioblastoma patient (CW1757) according T1-weighted MRI images] and PN3691 (VEC/BMI1) xenografted frozen brain tissue were fixed in 4% paraformaldehyde and immunolabeled using the primary antibodies against SOX2 (1:500, R & D system, AF2018), CD44 (1:100, BD Biosciences, 550392), vWF (1:500, Millipore, AB7356), CA9 (1:100, Genetex, GTX70020), H3K27Me3 (1:1000, Millipore, 07-689), H2AK119Ub (1:500, Cell signaling, #8240S), CD15 (1:100, Millipore, MAB4301), Ki67 (1:100, Dako, M7240), active CASPASE3 (1:100, Cell signaling, #9661S) and the secondary fluorescence-labeled antibodies. ..

    Chromatin Immunoprecipitation:

    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
    Article Snippet: .. Antibodies The following antibodies were used for Chromatin Immunoprecipitation (ChIP), RNA-binding protein Immunoprecipitation (RIP) or immunoblotting: anti-H3K27me3 (17–622, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-EZH2 (07–689, Millipore), anti-SUZ12 (3737, Cell Signaling Technology), anti-EED (17–663, Millipore), anti-GAPDH (M20006, Abmart). .. Immunoblotting Cells were lysed for 1 h at 4°C in ice-cold lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 150 mM NaCl, 0.5 mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% protease inhibitor cocktail [Sigma], 1 mM sodium orthovanadate, 1 mM NaF, 1% [vol/vol] Triton X-100 and 10% [vol/vol] glycerol).

    RNA Binding Assay:

    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
    Article Snippet: .. Antibodies The following antibodies were used for Chromatin Immunoprecipitation (ChIP), RNA-binding protein Immunoprecipitation (RIP) or immunoblotting: anti-H3K27me3 (17–622, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-EZH2 (07–689, Millipore), anti-SUZ12 (3737, Cell Signaling Technology), anti-EED (17–663, Millipore), anti-GAPDH (M20006, Abmart). .. Immunoblotting Cells were lysed for 1 h at 4°C in ice-cold lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 150 mM NaCl, 0.5 mM Ethylene Glycol Tetraacetic Acid (EGTA), 1% protease inhibitor cocktail [Sigma], 1 mM sodium orthovanadate, 1 mM NaF, 1% [vol/vol] Triton X-100 and 10% [vol/vol] glycerol).

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  • 91
    Millipore in vitro ezh2 inhibitor treatment
    Conditional deletion of the SET domain of <t>EZH2</t> in FOXP3 + Treg cells in mice (EZH2 Δ/Δ FOXP3 + or EZH2 Δ/+ FOXP3 + ) results in poor survival. A , exon map of mouse EZH2 indicating the EZH2 catalytic SET domain that was conditionally deleted by flanking LoxP insertion sites ( green arrowheads ). B , survival analysis of EZH2 Δ/Δ ( n = 27) or EZH2 Δ/+ ( n = 31) mice compared with WT ( n = 51) ( left panel ) and mutant mice distinguished by gender ( right panel ). The data are cumulative of over 10 litters, and all offspring are represented. C , representative images depicting the clinical appearance of experimental littermates and the size of FOXP3 wt EZH2 fl/fl (WT, 1 ), EZH2 wt/Δ FOXP3 + (EZH2 Δ/+ , 2 ) and EZH2 Δ/Δ FOXP3 + (EZH2 Δ/Δ , 3 ) pups and lack of ear, eye, and tail inflammation 21 days after birth. D, mean (S.E.) serum cytokine concentrations as measured by multiplex cytokine analysis. Data are from 15 biological replicates (unique animals) and represent independent experiments. Error bars denote S.E., n = 15 mice/group. Non-parametric unpaired t test was performed using Mann-Whitney t test, and p
    In Vitro Ezh2 Inhibitor Treatment, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    in vitro ezh2 inhibitor treatment - by Bioz Stars, 2020-09
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    93
    Millipore ezh2
    LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to <t>EZH2</t> and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p
    Ezh2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Millipore
    Average 93 stars, based on 75 article reviews
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    ezh2 - by Bioz Stars, 2020-09
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    Image Search Results


    Conditional deletion of the SET domain of EZH2 in FOXP3 + Treg cells in mice (EZH2 Δ/Δ FOXP3 + or EZH2 Δ/+ FOXP3 + ) results in poor survival. A , exon map of mouse EZH2 indicating the EZH2 catalytic SET domain that was conditionally deleted by flanking LoxP insertion sites ( green arrowheads ). B , survival analysis of EZH2 Δ/Δ ( n = 27) or EZH2 Δ/+ ( n = 31) mice compared with WT ( n = 51) ( left panel ) and mutant mice distinguished by gender ( right panel ). The data are cumulative of over 10 litters, and all offspring are represented. C , representative images depicting the clinical appearance of experimental littermates and the size of FOXP3 wt EZH2 fl/fl (WT, 1 ), EZH2 wt/Δ FOXP3 + (EZH2 Δ/+ , 2 ) and EZH2 Δ/Δ FOXP3 + (EZH2 Δ/Δ , 3 ) pups and lack of ear, eye, and tail inflammation 21 days after birth. D, mean (S.E.) serum cytokine concentrations as measured by multiplex cytokine analysis. Data are from 15 biological replicates (unique animals) and represent independent experiments. Error bars denote S.E., n = 15 mice/group. Non-parametric unpaired t test was performed using Mann-Whitney t test, and p

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Conditional deletion of the SET domain of EZH2 in FOXP3 + Treg cells in mice (EZH2 Δ/Δ FOXP3 + or EZH2 Δ/+ FOXP3 + ) results in poor survival. A , exon map of mouse EZH2 indicating the EZH2 catalytic SET domain that was conditionally deleted by flanking LoxP insertion sites ( green arrowheads ). B , survival analysis of EZH2 Δ/Δ ( n = 27) or EZH2 Δ/+ ( n = 31) mice compared with WT ( n = 51) ( left panel ) and mutant mice distinguished by gender ( right panel ). The data are cumulative of over 10 litters, and all offspring are represented. C , representative images depicting the clinical appearance of experimental littermates and the size of FOXP3 wt EZH2 fl/fl (WT, 1 ), EZH2 wt/Δ FOXP3 + (EZH2 Δ/+ , 2 ) and EZH2 Δ/Δ FOXP3 + (EZH2 Δ/Δ , 3 ) pups and lack of ear, eye, and tail inflammation 21 days after birth. D, mean (S.E.) serum cytokine concentrations as measured by multiplex cytokine analysis. Data are from 15 biological replicates (unique animals) and represent independent experiments. Error bars denote S.E., n = 15 mice/group. Non-parametric unpaired t test was performed using Mann-Whitney t test, and p

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Mouse Assay, Mutagenesis, Multiplex Assay, MANN-WHITNEY

    Pharmacologic inhibition of EZH2 reduces global H3K27me3 levels in vitro . A , immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD25 + cells cultured in the presence of the indicated inhibitors for 18 h. B , immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD62L + cells cultured under Treg-stimulating conditions (see “Experimental Procedures”) in the presence of the indicated inhibitors for 36 h. Note the toxicity of GSK126 and EPZ4638, as evident by reduction of Ponceau staining under these conditions.

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Pharmacologic inhibition of EZH2 reduces global H3K27me3 levels in vitro . A , immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD25 + cells cultured in the presence of the indicated inhibitors for 18 h. B , immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD62L + cells cultured under Treg-stimulating conditions (see “Experimental Procedures”) in the presence of the indicated inhibitors for 36 h. Note the toxicity of GSK126 and EPZ4638, as evident by reduction of Ponceau staining under these conditions.

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Inhibition, In Vitro, Cell Culture, Staining

    Pharmacologic inhibition of EZH2 results in heightened intestinal immune reactivity. A–D , demonstration of weight change ( A ), clinical disease activity index ( DAI , B ), colon length ( C ), and blinded histologic inflammatory index ( D ) in DZNep-treated animals versus sham-treated controls exposed to 2% DSS. E , representative colon histologic section demonstrating significant inflammation and ulceration. Statistical significance for weight change was determined using the Holm-Sidak method, with α = 5.000% ( p

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Pharmacologic inhibition of EZH2 results in heightened intestinal immune reactivity. A–D , demonstration of weight change ( A ), clinical disease activity index ( DAI , B ), colon length ( C ), and blinded histologic inflammatory index ( D ) in DZNep-treated animals versus sham-treated controls exposed to 2% DSS. E , representative colon histologic section demonstrating significant inflammation and ulceration. Statistical significance for weight change was determined using the Holm-Sidak method, with α = 5.000% ( p

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Inhibition, Activity Assay

    EZH2 Δ/Δ FOXP3 + mice develop end organ lymphoid infiltrates, either diffuse, nodular, or both. Shown are the heart, colon, small bowel, pancreas, lung, femoral-tibial joint, kidney, brain, liver, and mesenteric lymph node. Abnormal lymphoid infiltrate is evident in both a diffuse and nodular pattern ( arrows ). Four EZH2 wt/wt FOXP3 + and 7 EZH2 Δ/Δ FOXP3 + animals of both sexes were examined by histology. Representative images are shown. Histology was assessed by our participating pathologist blinded to study grouping.

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: EZH2 Δ/Δ FOXP3 + mice develop end organ lymphoid infiltrates, either diffuse, nodular, or both. Shown are the heart, colon, small bowel, pancreas, lung, femoral-tibial joint, kidney, brain, liver, and mesenteric lymph node. Abnormal lymphoid infiltrate is evident in both a diffuse and nodular pattern ( arrows ). Four EZH2 wt/wt FOXP3 + and 7 EZH2 Δ/Δ FOXP3 + animals of both sexes were examined by histology. Representative images are shown. Histology was assessed by our participating pathologist blinded to study grouping.

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Mouse Assay

    Tamoxifen-inducible loss of EZH2 results in heightened intestinal immune reactivity. A–D , demonstration of weight change ( A ), clinical disease activity index ( DAI , B ), colon length ( C ), and blinded histologic inflammatory index ( D ) in tamoxifen-treated CRE × EZH2 fl/fl animals versus tamoxifen-treated CRE EZH2 WT/WT controls exposed to 3% DSS. E , representative colon histologic section demonstrating significant inflammation and ulceration. Statistical significance for weight change was determined using the Holm-Sidak method, with α = 5.000% ( p

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Tamoxifen-inducible loss of EZH2 results in heightened intestinal immune reactivity. A–D , demonstration of weight change ( A ), clinical disease activity index ( DAI , B ), colon length ( C ), and blinded histologic inflammatory index ( D ) in tamoxifen-treated CRE × EZH2 fl/fl animals versus tamoxifen-treated CRE EZH2 WT/WT controls exposed to 3% DSS. E , representative colon histologic section demonstrating significant inflammation and ulceration. Statistical significance for weight change was determined using the Holm-Sidak method, with α = 5.000% ( p

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Activity Assay

    Up-regulation of differentially expressed FOXP3 and EZH2 targets indicates deregulation of a FOXP3-EZH2 pathway in Crohn's disease. A , heatmap of differential expression of 275 gene targets co-regulated by EZH2 and FOXP3. 187 of 275 gene targets were up-regulated. Shown is the rank order of ingenuity pathway application canonical pathways of up-regulated ( top bracket ) and down-regulated ( bottom bracket ) genes of patients compared with control subjects. B , ingenuity pathway analysis demonstrating the top enriched canonical pathways ( x axis), the enrichment of DEGs within each pathway ( y axis), and the status as up-regulation ( red ) or down-regulation ( green ). C , the ratio of TF (FOXP3, TBX21, or GATA3) and EZH2 common targets over non-common targets represented within the set of DEGs were compared using chi-squared test. Although the DEGs were significantly enriched for the common targets of FOXP3 and EZH2, there was no significant difference for the common targets of either TBX21 or GATA3 with EZH2.

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Up-regulation of differentially expressed FOXP3 and EZH2 targets indicates deregulation of a FOXP3-EZH2 pathway in Crohn's disease. A , heatmap of differential expression of 275 gene targets co-regulated by EZH2 and FOXP3. 187 of 275 gene targets were up-regulated. Shown is the rank order of ingenuity pathway application canonical pathways of up-regulated ( top bracket ) and down-regulated ( bottom bracket ) genes of patients compared with control subjects. B , ingenuity pathway analysis demonstrating the top enriched canonical pathways ( x axis), the enrichment of DEGs within each pathway ( y axis), and the status as up-regulation ( red ) or down-regulation ( green ). C , the ratio of TF (FOXP3, TBX21, or GATA3) and EZH2 common targets over non-common targets represented within the set of DEGs were compared using chi-squared test. Although the DEGs were significantly enriched for the common targets of FOXP3 and EZH2, there was no significant difference for the common targets of either TBX21 or GATA3 with EZH2.

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Expressing

    Pharmacologic inhibition of EZH2 reduces global H3K27me3 levels in vivo . A , representative immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD25 + cells, colon, and pancreas harvested from DZNep + 2% DSS-treated animals. B , quantification by densitometry of H3K27me3 immunoblot bands from isolated Treg cells in four independent experiments. For densitometry analysis, significance was determined using a non-parametric, unpaired t test of significance (Mann-Whitney). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: Pharmacologic inhibition of EZH2 reduces global H3K27me3 levels in vivo . A , representative immunoblot analysis of EZH2 and H3K27me3 levels in CD4 + CD25 + cells, colon, and pancreas harvested from DZNep + 2% DSS-treated animals. B , quantification by densitometry of H3K27me3 immunoblot bands from isolated Treg cells in four independent experiments. For densitometry analysis, significance was determined using a non-parametric, unpaired t test of significance (Mann-Whitney). *, p

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Inhibition, In Vivo, Isolation, MANN-WHITNEY

    EZH2 Δ/Δ FOXP3 + lymphocytes transform to a proinflammatory phenotype. A , the expression of cell surface markers in Treg cells (CD4+FOXP3+) measured by flow cytometry. Data are representative of three independent experiments ( n = 3 mice/experimental group). B , suppression assay measuring the degree of Treg-mediated suppression of CD4 + T responder cells (10 5 ) in response to anti-CD3 ± CD4 + CD25 + Treg titrations (10 5 , 0.5 × 10 5 , or 0.25 × 10 5 cells) from either EZH2 Δ/Δ , EZH2 Δ/+ , or WT mice. The rate of cell proliferation is directly proportional to titrated [ 3 H]thymidine uptake but inversely proportional to Treg-suppressive capability. The result demonstrated represents the mean (S.E.) of four independent experiments ( n = 4 biological replicates). Additionally, each independent experiment was performed in triplicate. Statistical significance was performed via non-parametric unpaired t test (Mann-Whitney t test). **, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD) *

    doi: 10.1074/jbc.M116.749663

    Figure Lengend Snippet: EZH2 Δ/Δ FOXP3 + lymphocytes transform to a proinflammatory phenotype. A , the expression of cell surface markers in Treg cells (CD4+FOXP3+) measured by flow cytometry. Data are representative of three independent experiments ( n = 3 mice/experimental group). B , suppression assay measuring the degree of Treg-mediated suppression of CD4 + T responder cells (10 5 ) in response to anti-CD3 ± CD4 + CD25 + Treg titrations (10 5 , 0.5 × 10 5 , or 0.25 × 10 5 cells) from either EZH2 Δ/Δ , EZH2 Δ/+ , or WT mice. The rate of cell proliferation is directly proportional to titrated [ 3 H]thymidine uptake but inversely proportional to Treg-suppressive capability. The result demonstrated represents the mean (S.E.) of four independent experiments ( n = 4 biological replicates). Additionally, each independent experiment was performed in triplicate. Statistical significance was performed via non-parametric unpaired t test (Mann-Whitney t test). **, p

    Article Snippet: For in vitro EZH2 inhibitor treatment, CD4+ CD25+ , CD4+ CD62L+ cells were cultured as described and treated with either vehicle or 5 μ m DZNep (Calbiochem), 5 μ m EPZ-6438 (Cayman Chemical), 5 μ m GSK503 (ApexBio), or 5 μ m GSK126 (Sellekchem).

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Suppression Assay, MANN-WHITNEY

    LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to EZH2 and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p

    Journal: Molecular Therapy

    Article Title: Long Noncoding RNA LINC00673 Is Activated by SP1 and Exerts Oncogenic Properties by Interacting with LSD1 and EZH2 in Gastric Cancer

    doi: 10.1016/j.ymthe.2017.01.017

    Figure Lengend Snippet: LINC00673 Suppresses LATS2 and KLF2 Transcription by Binding to EZH2 and LSD1 (A) FISH analysis of the location of LINC00673 (green) in the cytoplasm and nuclear fractions (blue) of BGC823 and SGC7901 cells. (B) qRT-PCR detection of the percentage of LINC00672, U1, and GAPDH in the cytoplasm and nuclear fractions of BGC823 and AGS cells. GAPDH and U1 were used as cytoplasmic and nuclear localization markers, respectively. (C) LINC00673 RNA levels in immunoprecipitates were determined by qRT-PCR. LINC00673 RNA expression levels are presented as fold enrichment values relative to IgG immunoprecipitates. (D) HuR, EZH2, LSD1, and STAU1 protein levels in immunoprecipitates with LINC00673 RNA were evaluated by western blots. Androgen receptor (AR) RNA was used as a positive control for HuR protein. The expression levels of HuR, EZH2 and LSD1 proteins are shown. (E) qRT-PCR analysis of CAMD4, LATS2, KLF2, and EZH2 expression in BGC823 and AGS cells after transfection with EZH2 or NC siRNA. (F) qRT-PCR analysis of CAMD4, LATS2, KLF2, and LSD1 expression in BGC823 and AGS cells after transfection with LSD1 or NC siRNA. (G) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the KLF2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. (H) ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the LATS2 promoter in BGC823 and AGS cells after transfection with LINC00673 or NC siRNA, with IgG used as a negative control. The mean values and SEs were calculated from triplicates of a representative experiment. *p

    Article Snippet: BGC-823 and AGS cells were lysed in complete RIP lysis buffer, and the extract was incubated with magnetic beads conjugated with antibodies that recognized EZH2, SUZ12, and LSD1 (and others) or control immunoglobulin G (IgG) (Millipore) for 6 hr at 4°C.

    Techniques: Binding Assay, Fluorescence In Situ Hybridization, Quantitative RT-PCR, RNA Expression, Western Blot, Positive Control, Expressing, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    EZH2 and BMI1 inversely correlate with variables associated with survival . (a) EZH2 and BMI1 distribution differ by grade. Mean EZH2 mRNA levels are -0.62 (n = 75), -0.21 (n = 101) and 0.37 (n = 119) for grades 1, 2 and 3, respectively (p

    Journal: Breast Cancer Research : BCR

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    doi: 10.1186/bcr2214

    Figure Lengend Snippet: EZH2 and BMI1 inversely correlate with variables associated with survival . (a) EZH2 and BMI1 distribution differ by grade. Mean EZH2 mRNA levels are -0.62 (n = 75), -0.21 (n = 101) and 0.37 (n = 119) for grades 1, 2 and 3, respectively (p

    Article Snippet: After antigen retrieval in citrate buffer, staining was performed using the Lab Vision Immunohistochemical Autostainer (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA) with primary antibodies against ER-alpha (1D5+6F11, dilution 1:50, Neomarkers (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA)), HER2 (3B5, dilution 1:3000), TP53 (DO-7, dilution 1:6000), EZH2 (AE25, dilution 1:10, gift from Kristian Helin) and BMI1 (F6, dilution 1:400, Millipore).

    Techniques:

    Model for the different oncogenic roles of EZH2 and BMI1 . (a) In normal development, genes such as the INK4a/ARF locus are bound by EZH2, marked by H3K27me3 and bound by BMI1. (b) On oncogene activation (indicated by lightning symbol) such as constitutive Ras or Myc expression, the INK4a/ARF locus becomes expressed as part of a tumour suppressive response. (c) Increased BMI1 expression results in more BMI1 protein binding to known Polycomb group (PcG)-target genes. This prevents activation of these genes even when signals are present that would normally activate these genes. (d) Increased EZH2 expression does not prevent INK4a/ARF activation (although this locus may still be inactivated by other mechanisms). In contrast, the role of EZH2 in tumourigenesis may be due to silencing of genes not normally targeted by PcG or due to methylation of non-histone proteins (indicated by diamond on protein 'X').

    Journal: Breast Cancer Research : BCR

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    doi: 10.1186/bcr2214

    Figure Lengend Snippet: Model for the different oncogenic roles of EZH2 and BMI1 . (a) In normal development, genes such as the INK4a/ARF locus are bound by EZH2, marked by H3K27me3 and bound by BMI1. (b) On oncogene activation (indicated by lightning symbol) such as constitutive Ras or Myc expression, the INK4a/ARF locus becomes expressed as part of a tumour suppressive response. (c) Increased BMI1 expression results in more BMI1 protein binding to known Polycomb group (PcG)-target genes. This prevents activation of these genes even when signals are present that would normally activate these genes. (d) Increased EZH2 expression does not prevent INK4a/ARF activation (although this locus may still be inactivated by other mechanisms). In contrast, the role of EZH2 in tumourigenesis may be due to silencing of genes not normally targeted by PcG or due to methylation of non-histone proteins (indicated by diamond on protein 'X').

    Article Snippet: After antigen retrieval in citrate buffer, staining was performed using the Lab Vision Immunohistochemical Autostainer (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA) with primary antibodies against ER-alpha (1D5+6F11, dilution 1:50, Neomarkers (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA)), HER2 (3B5, dilution 1:3000), TP53 (DO-7, dilution 1:6000), EZH2 (AE25, dilution 1:10, gift from Kristian Helin) and BMI1 (F6, dilution 1:400, Millipore).

    Techniques: Activation Assay, Expressing, Protein Binding, Methylation

    EZH2 and BMI1 are inversely correlated . (a). Univariate survival analyses by Kaplan-Meier plots and log rank tests. Patients were categorised by high or low expression of the different Polycomb group (PcG) members based on microarray data. (b) Relative risk of death evaluated by Cox regression modelling and continous PcG mRNA levels. Multivariate analysis is adjusted for tumour size, lymph node (LN) status and grade. (c) Relative risk of death based on continous EZH2 and BMI1 levels in LN-positive and LN-negative patients. P het = p value indicating heterogeneity of BMI1 levels across LN-positive and LN-negative patients. (d) Two-by-two table for categorical EZH2 and BMI1 levels. Odds ratio (OR) = 0.55 (95% confidence interval (CI) = 0.33 to 0.90) for high EZH2 among patients with high BMI1 compared with low BMI1 (chi-squared: p = 0.017).

    Journal: Breast Cancer Research : BCR

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    doi: 10.1186/bcr2214

    Figure Lengend Snippet: EZH2 and BMI1 are inversely correlated . (a). Univariate survival analyses by Kaplan-Meier plots and log rank tests. Patients were categorised by high or low expression of the different Polycomb group (PcG) members based on microarray data. (b) Relative risk of death evaluated by Cox regression modelling and continous PcG mRNA levels. Multivariate analysis is adjusted for tumour size, lymph node (LN) status and grade. (c) Relative risk of death based on continous EZH2 and BMI1 levels in LN-positive and LN-negative patients. P het = p value indicating heterogeneity of BMI1 levels across LN-positive and LN-negative patients. (d) Two-by-two table for categorical EZH2 and BMI1 levels. Odds ratio (OR) = 0.55 (95% confidence interval (CI) = 0.33 to 0.90) for high EZH2 among patients with high BMI1 compared with low BMI1 (chi-squared: p = 0.017).

    Article Snippet: After antigen retrieval in citrate buffer, staining was performed using the Lab Vision Immunohistochemical Autostainer (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA) with primary antibodies against ER-alpha (1D5+6F11, dilution 1:50, Neomarkers (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA)), HER2 (3B5, dilution 1:3000), TP53 (DO-7, dilution 1:6000), EZH2 (AE25, dilution 1:10, gift from Kristian Helin) and BMI1 (F6, dilution 1:400, Millipore).

    Techniques: Expressing, Microarray

    Correlation of EZH2 and BMI1 expression with molecular subtypes . (a) Mean mRNA levels of EZH2 and BMI1 in the different subgroups. The distribution of both EZH2 and BMI1 over the subgroups differ significantly, Kruskall-Wallis test. (b) Distribution of subgroups over categorised EZH2 and BMI1 levels. (c) Immunohistochemistry on EZH2 and BMI1 in normal breast tissue, an ErbB2-type and a luminal A-type tumour. Pictures were taken at 40× magnification (left panels) and a zoomed-in section is shown on the right of each image.

    Journal: Breast Cancer Research : BCR

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    doi: 10.1186/bcr2214

    Figure Lengend Snippet: Correlation of EZH2 and BMI1 expression with molecular subtypes . (a) Mean mRNA levels of EZH2 and BMI1 in the different subgroups. The distribution of both EZH2 and BMI1 over the subgroups differ significantly, Kruskall-Wallis test. (b) Distribution of subgroups over categorised EZH2 and BMI1 levels. (c) Immunohistochemistry on EZH2 and BMI1 in normal breast tissue, an ErbB2-type and a luminal A-type tumour. Pictures were taken at 40× magnification (left panels) and a zoomed-in section is shown on the right of each image.

    Article Snippet: After antigen retrieval in citrate buffer, staining was performed using the Lab Vision Immunohistochemical Autostainer (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA) with primary antibodies against ER-alpha (1D5+6F11, dilution 1:50, Neomarkers (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA)), HER2 (3B5, dilution 1:3000), TP53 (DO-7, dilution 1:6000), EZH2 (AE25, dilution 1:10, gift from Kristian Helin) and BMI1 (F6, dilution 1:400, Millipore).

    Techniques: Expressing, Immunohistochemistry

    Differential effect of BMI1 and EZH2 on INK4a/ARF locus and association with TP53 mutations . (a) Dotplot of INK4a/ARF (CDKN2A) expression versus BMI1 and EZH2 expression. INK4a/ARF expression is not found in tumours with high BMI1 mRNA, whereas it does occur in tumours with high EZH2 (red circle). (b) Distribution of patients with positive TP53 staining (mutated TP53) differs by categorised EZH2 and BMI1 levels. TP53 immunohistochemistry (IHC) data was obtained from 273 patients and the TP53 sequence was analysed in 204 tumours. The distribution of mutant TP53 shows a similar pattern (TP53 mutation). (c) Logistic regression analysis of TP53 IHC and sequence status on EZH2 and BMI1 levels. CI = confidence interval, OR = odds ratio.

    Journal: Breast Cancer Research : BCR

    Article Title: EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

    doi: 10.1186/bcr2214

    Figure Lengend Snippet: Differential effect of BMI1 and EZH2 on INK4a/ARF locus and association with TP53 mutations . (a) Dotplot of INK4a/ARF (CDKN2A) expression versus BMI1 and EZH2 expression. INK4a/ARF expression is not found in tumours with high BMI1 mRNA, whereas it does occur in tumours with high EZH2 (red circle). (b) Distribution of patients with positive TP53 staining (mutated TP53) differs by categorised EZH2 and BMI1 levels. TP53 immunohistochemistry (IHC) data was obtained from 273 patients and the TP53 sequence was analysed in 204 tumours. The distribution of mutant TP53 shows a similar pattern (TP53 mutation). (c) Logistic regression analysis of TP53 IHC and sequence status on EZH2 and BMI1 levels. CI = confidence interval, OR = odds ratio.

    Article Snippet: After antigen retrieval in citrate buffer, staining was performed using the Lab Vision Immunohistochemical Autostainer (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA) with primary antibodies against ER-alpha (1D5+6F11, dilution 1:50, Neomarkers (Lab Vision Products Thermo Fisher Scientific, Fremont, CA, USA)), HER2 (3B5, dilution 1:3000), TP53 (DO-7, dilution 1:6000), EZH2 (AE25, dilution 1:10, gift from Kristian Helin) and BMI1 (F6, dilution 1:400, Millipore).

    Techniques: Expressing, Staining, Immunohistochemistry, Sequencing, Mutagenesis

    LINC01133 interacted with EZH2 and LSD1, and silence KLF2, P21 and E-cadherin expression A. Relative LINC01133 levels in cell cytoplasm or Nucleus of NSCLC cell lines were detected by qPCR. B. RNA levels in immunoprecipitates were determined by qPCR. Expression levels of LINC01133 RNA were presented as fold enrichment relative to IgG immunoprecipitates. C. Protein levels in immunoprecipitates were determined by western blot. Expression levels of EZH2 and LSD1 protein were presented. D. The levels of KLF2, P21, PTEN, LATS2, RRAD, ASPP2 and E-cadherin mRNA were determined by qPCR when knockdown of LINC01133. E. The KLF2, P21 and E-cadherin protein levels were determined by western blot in LINC01133 knockdown A549 and PC9 cells. F. The E-cadherin protein levels were determined by immunofluorescence analysis in LINC01133 knockdown A549 cells. G. The KLF2, P21 and E-cadherin expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in A549 and PC9 cells. H, I. ChIP–qPCR of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding in the KLF2, P21 or E-cadherin promoter in A549 and PC9 cells, and IgG as a negative control. The mean values and s.d. were calculated from triplicates of a representative experiment.

    Journal: Oncotarget

    Article Title: Long non-coding RNA LINC01133 represses KLF2, P21 and E-cadherin transcription through binding with EZH2, LSD1 in non small cell lung cancer

    doi: 10.18632/oncotarget.7077

    Figure Lengend Snippet: LINC01133 interacted with EZH2 and LSD1, and silence KLF2, P21 and E-cadherin expression A. Relative LINC01133 levels in cell cytoplasm or Nucleus of NSCLC cell lines were detected by qPCR. B. RNA levels in immunoprecipitates were determined by qPCR. Expression levels of LINC01133 RNA were presented as fold enrichment relative to IgG immunoprecipitates. C. Protein levels in immunoprecipitates were determined by western blot. Expression levels of EZH2 and LSD1 protein were presented. D. The levels of KLF2, P21, PTEN, LATS2, RRAD, ASPP2 and E-cadherin mRNA were determined by qPCR when knockdown of LINC01133. E. The KLF2, P21 and E-cadherin protein levels were determined by western blot in LINC01133 knockdown A549 and PC9 cells. F. The E-cadherin protein levels were determined by immunofluorescence analysis in LINC01133 knockdown A549 cells. G. The KLF2, P21 and E-cadherin expression levels were determined by qPCR when knockdown of EZH2 or LSD1 in A549 and PC9 cells. H, I. ChIP–qPCR of EZH2 and LSD1 occupancy, H3K27me3 and H3K4me2 binding in the KLF2, P21 or E-cadherin promoter in A549 and PC9 cells, and IgG as a negative control. The mean values and s.d. were calculated from triplicates of a representative experiment.

    Article Snippet: The supernatants were incubated with protein A/G Sepharose beads coated with antibodies that recognized EZH2, SUZ12, LSD1, SNRNP70 or with control IgG (millipore) for 6hr at 4°C.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Chromatin Immunoprecipitation, Binding Assay, Negative Control