ezh2  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2
    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for <t>EZH2,</t> EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin"

    Article Title: Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin

    Journal: Journal of Cancer

    doi: 10.7150/jca.63234

    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    Figure Legend Snippet: Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).

    Techniques Used: In Vitro, Western Blot, Immunohistochemistry, Staining

    5246s  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc 5246s
    5246s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5246s/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    5246s - by Bioz Stars, 2023-02
    86/100 stars

    Images

    ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2
    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for <t>EZH2,</t> EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin"

    Article Title: Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin

    Journal: Journal of Cancer

    doi: 10.7150/jca.63234

    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    Figure Legend Snippet: Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).

    Techniques Used: In Vitro, Western Blot, Immunohistochemistry, Staining

    rabbit monoclonal anti ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc rabbit monoclonal anti ezh2
    FOXP3 interacts with <t>EZH2</t> in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.
    Rabbit Monoclonal Anti Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal anti ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation"

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2018.08.009

    FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.
    Figure Legend Snippet: FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

    Techniques Used: Isolation, Derivative Assay, Ligation, Amplification, Immunoprecipitation, Expressing, Activation Assay

    FOXP3 constitutively interacts with the PRC2 complex. HEK293T cells transfected with plasmids encoding either EZH2 (myc-tagged EZH2), FOXP3 (His-tagged FOXP3), or both for 48 hours were subjected to PLA or co-immunoprecipitation using the indicated primary antibodies. ( A ) Representative PLA images of cells from 3 independent experiments; first 3 rows are negative control experiments and green signals indicate a FOXP3–EZH2 interaction. Scale bar : 20 μm. ( B ) Quantitation of nuclear PLA signals in images from panel A ; n = number of cells imaged. *** P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Whole-cell lysates from HEK293T cells transfected with plasmids encoding His–FOXP3 and myc–DDK–tagged EZH2 were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody, immunoblotted for His–FOXP3 and myc–EZH2 with FOXP3 and myc antibodies. Input shows protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( D ) Cells from panel C were subjected to PLA using His antibody (negative control) or both His and myc antibodies. Red signals indicate FOXP3–EZH2 interaction; data are representative of 3 independent experiments. Scale bar : 20 μm. ( E ) Quantitation from 3 independent experiments of nuclear PLA signals in images from <xref ref-type=Figure 1 D . ∗∗∗ P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test). n = number of cells imaged. ( F and G ) Cell lysates from cell lines transfected with the indicated plasmids (FOXP3 and EZH2 in HEK293T cells or FOXP3 alone in Jurkat T cells) were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody; immunoblotted for His–FOXP3, myc–EZH2, and the other PRC2 subunits SUZ12 and EED with the indicated antibodies. Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole. " title="... complex. HEK293T cells transfected with plasmids encoding either EZH2 (myc-tagged EZH2), FOXP3 (His-tagged FOXP3), or both for ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: FOXP3 constitutively interacts with the PRC2 complex. HEK293T cells transfected with plasmids encoding either EZH2 (myc-tagged EZH2), FOXP3 (His-tagged FOXP3), or both for 48 hours were subjected to PLA or co-immunoprecipitation using the indicated primary antibodies. ( A ) Representative PLA images of cells from 3 independent experiments; first 3 rows are negative control experiments and green signals indicate a FOXP3–EZH2 interaction. Scale bar : 20 μm. ( B ) Quantitation of nuclear PLA signals in images from panel A ; n = number of cells imaged. *** P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Whole-cell lysates from HEK293T cells transfected with plasmids encoding His–FOXP3 and myc–DDK–tagged EZH2 were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody, immunoblotted for His–FOXP3 and myc–EZH2 with FOXP3 and myc antibodies. Input shows protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( D ) Cells from panel C were subjected to PLA using His antibody (negative control) or both His and myc antibodies. Red signals indicate FOXP3–EZH2 interaction; data are representative of 3 independent experiments. Scale bar : 20 μm. ( E ) Quantitation from 3 independent experiments of nuclear PLA signals in images from Figure 1 D . ∗∗∗ P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test). n = number of cells imaged. ( F and G ) Cell lysates from cell lines transfected with the indicated plasmids (FOXP3 and EZH2 in HEK293T cells or FOXP3 alone in Jurkat T cells) were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody; immunoblotted for His–FOXP3, myc–EZH2, and the other PRC2 subunits SUZ12 and EED with the indicated antibodies. Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

    Techniques Used: Transfection, Immunoprecipitation, Negative Control, Quantitation Assay, Expressing

    FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P < .0001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( C ) Jurkat cells expressing vector or FOXP3 (WT or mutants) plus luciferase plasmids were treated with DMSO or PMA/ionomycin as in panel B . Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. *** P < .001 (1-way analysis of variance + Bonferroni test). ( D and E ) Whole-cell lysates from HEK293T cells co-expressing myc-tagged EZH2 and His–FOXP3 WT or mutants were subjected to FOXP3 immunoprecipitation, immunoblotted for His–FOXP3 and myc–EZH2 using the indicated antibodies. Red arrows and gray arrows emphasize C232 mutants within FOXP3. Data shown are representative of 3 independent experiments. ( F ) Jurkat cells expressing empty vector or His–FOXP3 (WT or C232 mutants) and indicated luciferase plasmids were treated with 0.4% DMSO or PMA/ionomycin. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates; *** P < .001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( G ) Chromatin from Jurkat cells expressing FOXP3–WT or –C232Δ were incubated with IgG control or anti-H3K27me3 antibody, polymerase chain reaction (PCR) for the IFNγ promoter was performed to assess the presence of H3K27me3 repressive mark. Data shown are representative of 3 independent experiments. ( H ) Jurkat cells overexpressing empty His-vector plasmid (control), His–FOXP3 WT or His–FOXP3 mutants (C232G and C232Δ) were treated with 0.2% DMSO ( top row ) or PMA/ionomycin ( bottom row ) for 12 hours. Cells were permeabilized and then stained with fluorescently conjugated IL2 and His antibodies against intracellular IL2 and His-tagged FOXP3. Dot plot in quadrant 2 (Q2) depicts the frequency, in percentage, of IL2 and His co-expressing cells as measured by flow cytometry. Data shown are representative of 3 independent experiments.
    Figure Legend Snippet: FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P < .0001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( C ) Jurkat cells expressing vector or FOXP3 (WT or mutants) plus luciferase plasmids were treated with DMSO or PMA/ionomycin as in panel B . Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. *** P < .001 (1-way analysis of variance + Bonferroni test). ( D and E ) Whole-cell lysates from HEK293T cells co-expressing myc-tagged EZH2 and His–FOXP3 WT or mutants were subjected to FOXP3 immunoprecipitation, immunoblotted for His–FOXP3 and myc–EZH2 using the indicated antibodies. Red arrows and gray arrows emphasize C232 mutants within FOXP3. Data shown are representative of 3 independent experiments. ( F ) Jurkat cells expressing empty vector or His–FOXP3 (WT or C232 mutants) and indicated luciferase plasmids were treated with 0.4% DMSO or PMA/ionomycin. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates; *** P < .001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( G ) Chromatin from Jurkat cells expressing FOXP3–WT or –C232Δ were incubated with IgG control or anti-H3K27me3 antibody, polymerase chain reaction (PCR) for the IFNγ promoter was performed to assess the presence of H3K27me3 repressive mark. Data shown are representative of 3 independent experiments. ( H ) Jurkat cells overexpressing empty His-vector plasmid (control), His–FOXP3 WT or His–FOXP3 mutants (C232G and C232Δ) were treated with 0.2% DMSO ( top row ) or PMA/ionomycin ( bottom row ) for 12 hours. Cells were permeabilized and then stained with fluorescently conjugated IL2 and His antibodies against intracellular IL2 and His-tagged FOXP3. Dot plot in quadrant 2 (Q2) depicts the frequency, in percentage, of IL2 and His co-expressing cells as measured by flow cytometry. Data shown are representative of 3 independent experiments.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Immunoprecipitation, Incubation, Polymerase Chain Reaction, Staining, Flow Cytometry

    IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P < .001, ∗ P < .05. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Quantitation of red fluorescent intensities displayed by representative cells shown in panel A . Data are representative of 3 independent experiments. ( D ) Representative confocal microscopic PLA images of intestinal CD4 + T cells from 3 CD patients showing reduced FOXP3–EZH2 complexes (white signals) in comparison with non-CD control cells. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Scale bar : 2–5 μm. Simple endoscopic scores for CD patients were as follows: patient 1, 18; patient 2 (on 30 mg of prednisone), 10; and patient 3 (on 8 mg of budesonide 1 time daily), 8. ( E ) Lamina propria CD4 + T cells from panel D were stained with fluorescently conjugated FOXP3 antibody and subjected to flow cytometric analysis. Histogram overlay compares FOXP3 expression in isolated CD4 + T cells (control vs CD patients 2 and 3). ( F ) Quantitation of nuclear PLA signals/CD4 + T cells from individual CD patients (patients 1, 2, or 3) vs non-CD CD4 + T cells. n = number of cells imaged. Red horizontal bars denote means ± SEM. **** P < .001; NS, 1-way analysis of variance + Bonferroni test. ( G ) Quantitation of nuclear PLA signals/CD4 + T cells from all 3 CD patients vs non-CD control as shown in panel F . n = number of cells imaged. **** P < .0001. Red horizontal bars indicate means ± SEM (Student t test). ( H ) PBMC-derived human Tregs were treated with DMSO, IL6 (50 ng/mL), or IL6 (50 ng/mL) plus 10 μmol/L ruxolitinib (ruxo.) for 2 hours in serum-free media as in panel A , and then permeabilized and stained for FOXP3 or EZH2 with fluorochrome-conjugated antibody or primary antibody, respectively. Dot plots show the frequency, in percentage, of cells expressing FOXP3 ( top row : quadrant 7 [Q7]) or EZH2 ( bottom row : quadrant 3 [Q3]) as measured by flow cytometric analysis. For negative controls, IgG isotype or fluorescently conjugated secondary antibody were used to stain cells. ( I ) Histograms depict FOXP3 or EZH2 expression in cells from panel H . Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; FI, fluorescent intensity; SSC, side scatter.
    Figure Legend Snippet: IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P < .001, ∗ P < .05. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Quantitation of red fluorescent intensities displayed by representative cells shown in panel A . Data are representative of 3 independent experiments. ( D ) Representative confocal microscopic PLA images of intestinal CD4 + T cells from 3 CD patients showing reduced FOXP3–EZH2 complexes (white signals) in comparison with non-CD control cells. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Scale bar : 2–5 μm. Simple endoscopic scores for CD patients were as follows: patient 1, 18; patient 2 (on 30 mg of prednisone), 10; and patient 3 (on 8 mg of budesonide 1 time daily), 8. ( E ) Lamina propria CD4 + T cells from panel D were stained with fluorescently conjugated FOXP3 antibody and subjected to flow cytometric analysis. Histogram overlay compares FOXP3 expression in isolated CD4 + T cells (control vs CD patients 2 and 3). ( F ) Quantitation of nuclear PLA signals/CD4 + T cells from individual CD patients (patients 1, 2, or 3) vs non-CD CD4 + T cells. n = number of cells imaged. Red horizontal bars denote means ± SEM. **** P < .001; NS, 1-way analysis of variance + Bonferroni test. ( G ) Quantitation of nuclear PLA signals/CD4 + T cells from all 3 CD patients vs non-CD control as shown in panel F . n = number of cells imaged. **** P < .0001. Red horizontal bars indicate means ± SEM (Student t test). ( H ) PBMC-derived human Tregs were treated with DMSO, IL6 (50 ng/mL), or IL6 (50 ng/mL) plus 10 μmol/L ruxolitinib (ruxo.) for 2 hours in serum-free media as in panel A , and then permeabilized and stained for FOXP3 or EZH2 with fluorochrome-conjugated antibody or primary antibody, respectively. Dot plots show the frequency, in percentage, of cells expressing FOXP3 ( top row : quadrant 7 [Q7]) or EZH2 ( bottom row : quadrant 3 [Q3]) as measured by flow cytometric analysis. For negative controls, IgG isotype or fluorescently conjugated secondary antibody were used to stain cells. ( I ) Histograms depict FOXP3 or EZH2 expression in cells from panel H . Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; FI, fluorescent intensity; SSC, side scatter.

    Techniques Used: Inhibition, Isolation, Derivative Assay, Quantitation Assay, Staining, Expressing

    IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments using cells from PBMC donors. ( E and F ) PBMC-derived human Tregs were treated with IL6 or both IL6 and JAK1/2 inhibitor ruxolitinib for the indicated time points. PLA and confocal microscopic imaging were performed to detect and visualize phosphorylated (p) residues (p-Y705 and p-S727) on STAT3 (red signals) by staining cells with the indicated antibodies. Representative images from 3 independent experiments are shown from 3 different donors. White dotted lines mark the cytoplasm of cells while 4′,6-diamidino-2-phenylindole (DAPI) stains the nuclei blue. Red PLA signals indicate phosphorylated tyrosine residue (Y) 705 on STAT3 in cells stained with both STAT3 and p-STAT3-Y705 antibodies or phosphorylated serine residue (S) 727 on STAT3 in cells stained with both STAT3 and p-STAT3-S727 antibodies. Scale bar : 5 μm. ( G and H ) Quantitation of nuclear PLA signals from images in panels E and F , respectively. n = number of cells imaged. Red horizontal bars denote means ± SEM. ** P < .01 and *** P < .001 (1-way analysis of variance + Bonferroni test) from 3 independent experiments across 3 different donors.
    Figure Legend Snippet: IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments using cells from PBMC donors. ( E and F ) PBMC-derived human Tregs were treated with IL6 or both IL6 and JAK1/2 inhibitor ruxolitinib for the indicated time points. PLA and confocal microscopic imaging were performed to detect and visualize phosphorylated (p) residues (p-Y705 and p-S727) on STAT3 (red signals) by staining cells with the indicated antibodies. Representative images from 3 independent experiments are shown from 3 different donors. White dotted lines mark the cytoplasm of cells while 4′,6-diamidino-2-phenylindole (DAPI) stains the nuclei blue. Red PLA signals indicate phosphorylated tyrosine residue (Y) 705 on STAT3 in cells stained with both STAT3 and p-STAT3-Y705 antibodies or phosphorylated serine residue (S) 727 on STAT3 in cells stained with both STAT3 and p-STAT3-S727 antibodies. Scale bar : 5 μm. ( G and H ) Quantitation of nuclear PLA signals from images in panels E and F , respectively. n = number of cells imaged. Red horizontal bars denote means ± SEM. ** P < .01 and *** P < .001 (1-way analysis of variance + Bonferroni test) from 3 independent experiments across 3 different donors.

    Techniques Used: Activation Assay, Expressing, Immunoprecipitation, Staining, Quantitation Assay, Derivative Assay, Imaging

    ezh2 5246 antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2 5246 antibody
    Paired primer sequences used in qRT-PCR
    Ezh2 5246 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 5246 antibody/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 5246 antibody - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways"

    Article Title: lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways

    Journal: Cell Journal (Yakhteh)

    doi: 10.22074/cellj.2021.7010

    Paired primer sequences used in qRT-PCR
    Figure Legend Snippet: Paired primer sequences used in qRT-PCR

    Techniques Used:

    Long non-coding RNAs (lncRNAs)-plasmacytoma variant translocation 1( PVT1 ) directly interacted with enhancer of zeste homolog 2 (EZH2) in non-small cell lung cancer (NSCLC). A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in the NSCLC and adjacent normal tissues (n=30). B. qRT-PCR and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in NSCLC cell lines compared to the normal lung epithelial cells, BEAS-2B. C. Relationship between lncRNA PVT1 and EZH2 verified in A549 cells by RNA pull-down and western blot assays using a biotin-labeled probe of PVT1 (bio-PVT1). D. RNA pull-down and western blot assays used to verify the PVT1 effect on EZH2 level after PVT1 knockdown by treating with si-lncPVT1 or si-NC in A549 cells. Data are mean ± SD of three independent experiments. *; P<0.05, **; P<0.01, ***; P<0.001 compared to the adjacent group or MRC5 group. si-NC; siRNA negative control, Bio-NC; Biotin-labeled negative control, Pull; Pull-down group, and SD; Standard deviation.
    Figure Legend Snippet: Long non-coding RNAs (lncRNAs)-plasmacytoma variant translocation 1( PVT1 ) directly interacted with enhancer of zeste homolog 2 (EZH2) in non-small cell lung cancer (NSCLC). A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in the NSCLC and adjacent normal tissues (n=30). B. qRT-PCR and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in NSCLC cell lines compared to the normal lung epithelial cells, BEAS-2B. C. Relationship between lncRNA PVT1 and EZH2 verified in A549 cells by RNA pull-down and western blot assays using a biotin-labeled probe of PVT1 (bio-PVT1). D. RNA pull-down and western blot assays used to verify the PVT1 effect on EZH2 level after PVT1 knockdown by treating with si-lncPVT1 or si-NC in A549 cells. Data are mean ± SD of three independent experiments. *; P<0.05, **; P<0.01, ***; P<0.001 compared to the adjacent group or MRC5 group. si-NC; siRNA negative control, Bio-NC; Biotin-labeled negative control, Pull; Pull-down group, and SD; Standard deviation.

    Techniques Used: Variant Assay, Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Labeling, Negative Control, Standard Deviation

    pcDNA-PVT1 vector for overexpression (PVT1) regulated expression of yes-associated protein 1 (YAP1) through enhancer of zeste homolog 2 (EZH2) -mediated microRNA-497(miR-497) promoter methylation. A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for the expression levels of EZH2 , miR-497 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with si-EZH2 or si-NC. B. Western blot analysis for the protein levels of EZH2 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with EZH2 siRNA for knockdown (si-EZH2) or si-NC. C. ChIP–qPCR of EZH2 occupancy and H3K27me3 binding in the miR-497 promoter in H1299 cells with or without PVT1 overexpression. a, b and c represented respectively three pairs of amplification primers for miR-497 promoter; and d and e represented respectively two pairs of amplification primers for U6 promoter as internal references. D. MSP analysis for the methylation level of miR-497 promoter in A549 and H1299 cells treated with si-EZH2 or 5-Aza-dC. E. qRT-PCR analysis for the expression levels of miR-497 and YAP1 in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. F. Western blot analysis for the protein levels of LAST2 and YAP1 phosphorylation in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. Data are mean ± SD of three independent experiments, *; P<0.05, **; P<0.01 compared between siRNA negative control (si-NC) and si-EZH2 groups or compared between groups as shown with a horizontal line.5-aza2-deoxycytidine;A549 and H1299; Two of human lung carcinoma cell lines, ChIP; Chromatin immunoprecipitation, MSP; Methylation-specific polymerase chain reaction, LAST2; Large tumor suppressor kinase 2 protein, and SD; Standard deviation.
    Figure Legend Snippet: pcDNA-PVT1 vector for overexpression (PVT1) regulated expression of yes-associated protein 1 (YAP1) through enhancer of zeste homolog 2 (EZH2) -mediated microRNA-497(miR-497) promoter methylation. A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for the expression levels of EZH2 , miR-497 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with si-EZH2 or si-NC. B. Western blot analysis for the protein levels of EZH2 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with EZH2 siRNA for knockdown (si-EZH2) or si-NC. C. ChIP–qPCR of EZH2 occupancy and H3K27me3 binding in the miR-497 promoter in H1299 cells with or without PVT1 overexpression. a, b and c represented respectively three pairs of amplification primers for miR-497 promoter; and d and e represented respectively two pairs of amplification primers for U6 promoter as internal references. D. MSP analysis for the methylation level of miR-497 promoter in A549 and H1299 cells treated with si-EZH2 or 5-Aza-dC. E. qRT-PCR analysis for the expression levels of miR-497 and YAP1 in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. F. Western blot analysis for the protein levels of LAST2 and YAP1 phosphorylation in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. Data are mean ± SD of three independent experiments, *; P<0.05, **; P<0.01 compared between siRNA negative control (si-NC) and si-EZH2 groups or compared between groups as shown with a horizontal line.5-aza2-deoxycytidine;A549 and H1299; Two of human lung carcinoma cell lines, ChIP; Chromatin immunoprecipitation, MSP; Methylation-specific polymerase chain reaction, LAST2; Large tumor suppressor kinase 2 protein, and SD; Standard deviation.

    Techniques Used: Plasmid Preparation, Over Expression, Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Binding Assay, Amplification, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation

    anti ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc anti ezh2
    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of <t>EZH2,</t> SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.
    Anti Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer"

    Article Title: HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer

    Journal: Journal of Cancer

    doi: 10.7150/jca.59740

    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.
    Figure Legend Snippet: Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.

    Techniques Used: Amplification, Chromatin Immunoprecipitation, Western Blot

    anti ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc anti ezh2
    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of <t>EZH2,</t> SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.
    Anti Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer"

    Article Title: HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer

    Journal: Journal of Cancer

    doi: 10.7150/jca.59740

    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.
    Figure Legend Snippet: Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.

    Techniques Used: Amplification, Chromatin Immunoprecipitation, Western Blot

    ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2
    FOXO1 gene is a repression target of <t>EZH2.</t> (A) EZH2 ChIP-on-chip assay reveals murine Ezh2 binds to the Foxo1 promoter in mouse embryo stem cells. (B) Screen shot of the UCSC genome browser showing ChIP-seq (reported previously , ) signal profiles of EZH2 binding in the FOXO1 gene locus in different human cell lines. (C) ChIP-qPCR analysis of EZH2 occupancy in the FOXO1 promoter in both C4-2 and 22Rv1 prostate cancer cell lines. (D) ChIP-qPCR analysis of H3K27me3 enrichment in the FOXO1 promoter in prostate cancer cell line C4-2 and 22Rv1 cells. (E) RT-qPCR analysis of FOXO1 mRNA expression in C4-2 and 22Rv1 cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. RT-PCR for GAPDH was utilized as an internal control. (F) Western blot analysis of FOXO1 and EZH2 proteins in C4-2 and 22Rv1cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. ERK2 was used as a loading control. (G, H) RT-qPCR (G) and western blot (H) analysis of FOXO1 mRNA and protein expression in C4-2 and 22Rv1 cells transfected with empty vector or EZH1-specific sgRNA and selected with puromycin for one week. RT-qPCR for GAPDH was utilized as an internal control. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "Overcoming EZH2 Inhibitor Resistance by Taxane in PTEN-Mutated Cancer"

    Article Title: Overcoming EZH2 Inhibitor Resistance by Taxane in PTEN-Mutated Cancer

    Journal: Theranostics

    doi: 10.7150/thno.34700

    FOXO1 gene is a repression target of EZH2. (A) EZH2 ChIP-on-chip assay reveals murine Ezh2 binds to the Foxo1 promoter in mouse embryo stem cells. (B) Screen shot of the UCSC genome browser showing ChIP-seq (reported previously , ) signal profiles of EZH2 binding in the FOXO1 gene locus in different human cell lines. (C) ChIP-qPCR analysis of EZH2 occupancy in the FOXO1 promoter in both C4-2 and 22Rv1 prostate cancer cell lines. (D) ChIP-qPCR analysis of H3K27me3 enrichment in the FOXO1 promoter in prostate cancer cell line C4-2 and 22Rv1 cells. (E) RT-qPCR analysis of FOXO1 mRNA expression in C4-2 and 22Rv1 cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. RT-PCR for GAPDH was utilized as an internal control. (F) Western blot analysis of FOXO1 and EZH2 proteins in C4-2 and 22Rv1cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. ERK2 was used as a loading control. (G, H) RT-qPCR (G) and western blot (H) analysis of FOXO1 mRNA and protein expression in C4-2 and 22Rv1 cells transfected with empty vector or EZH1-specific sgRNA and selected with puromycin for one week. RT-qPCR for GAPDH was utilized as an internal control. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Figure Legend Snippet: FOXO1 gene is a repression target of EZH2. (A) EZH2 ChIP-on-chip assay reveals murine Ezh2 binds to the Foxo1 promoter in mouse embryo stem cells. (B) Screen shot of the UCSC genome browser showing ChIP-seq (reported previously , ) signal profiles of EZH2 binding in the FOXO1 gene locus in different human cell lines. (C) ChIP-qPCR analysis of EZH2 occupancy in the FOXO1 promoter in both C4-2 and 22Rv1 prostate cancer cell lines. (D) ChIP-qPCR analysis of H3K27me3 enrichment in the FOXO1 promoter in prostate cancer cell line C4-2 and 22Rv1 cells. (E) RT-qPCR analysis of FOXO1 mRNA expression in C4-2 and 22Rv1 cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. RT-PCR for GAPDH was utilized as an internal control. (F) Western blot analysis of FOXO1 and EZH2 proteins in C4-2 and 22Rv1cells transfected with non-specific (NS) control or a pool of EZH2-specific siRNA for 48 h. ERK2 was used as a loading control. (G, H) RT-qPCR (G) and western blot (H) analysis of FOXO1 mRNA and protein expression in C4-2 and 22Rv1 cells transfected with empty vector or EZH1-specific sgRNA and selected with puromycin for one week. RT-qPCR for GAPDH was utilized as an internal control. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.

    Techniques Used: ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Two Tailed Test

    EZH2 expression inversely correlates with FOXO1 level in prostate cancer patient specimens. (A) Correlation analysis of EZH2 and FOXO1 mRNA expression in a cohort of primary (n = 59) and metastatic (n = 35) prostate cancer specimens reported previously . (B) Representative images of IHC staining of EZH2 and FOXO1 antibodies on prostate cancer patient specimens (n = 42). Scale bar in 10 X fields: 100 μm; Scale bar in 40 X fields: 20 μm. (C) Correlation analysis of IHC staining of EZH2 and FOXO1 proteins in prostate cancer patient specimens (n = 42). (D) Heat map showing IHC score (see calculation details in Materials and Methods) of EZH2 and FOXO1 protein staining on prostate cancer tissues.
    Figure Legend Snippet: EZH2 expression inversely correlates with FOXO1 level in prostate cancer patient specimens. (A) Correlation analysis of EZH2 and FOXO1 mRNA expression in a cohort of primary (n = 59) and metastatic (n = 35) prostate cancer specimens reported previously . (B) Representative images of IHC staining of EZH2 and FOXO1 antibodies on prostate cancer patient specimens (n = 42). Scale bar in 10 X fields: 100 μm; Scale bar in 40 X fields: 20 μm. (C) Correlation analysis of IHC staining of EZH2 and FOXO1 proteins in prostate cancer patient specimens (n = 42). (D) Heat map showing IHC score (see calculation details in Materials and Methods) of EZH2 and FOXO1 protein staining on prostate cancer tissues.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    The methyltransferase activity of EZH2 and other core components of the PRC2 complex are important for EZH2-mediated repression of FOXO1. (A, B) C4-2 and 22Rv1 cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis (A) and RT-qPCR (B) . ERK2 was used as a loading control in western blot. GAPDH was utilized as an internal control in RT-PCR. (C, D) C4-2 and 22Rv1 cells were infected with lentivirus expressing non-specific shRNA (shNS) or SUZ12-specific shRNAs for 48 h and harvested for western blot analysis (C) and RT-qPCR (D) . (E, F) C4-2 and 22Rv1 cells were infected with lentivirus expressing non-specific shRNA (shNS) or EED-specific shRNAs for 48 h and harvested for western blot analysis ( E ) and RT-qPCR ( F ). Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Figure Legend Snippet: The methyltransferase activity of EZH2 and other core components of the PRC2 complex are important for EZH2-mediated repression of FOXO1. (A, B) C4-2 and 22Rv1 cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis (A) and RT-qPCR (B) . ERK2 was used as a loading control in western blot. GAPDH was utilized as an internal control in RT-PCR. (C, D) C4-2 and 22Rv1 cells were infected with lentivirus expressing non-specific shRNA (shNS) or SUZ12-specific shRNAs for 48 h and harvested for western blot analysis (C) and RT-qPCR (D) . (E, F) C4-2 and 22Rv1 cells were infected with lentivirus expressing non-specific shRNA (shNS) or EED-specific shRNAs for 48 h and harvested for western blot analysis ( E ) and RT-qPCR ( F ). Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.

    Techniques Used: Activity Assay, Transfection, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, shRNA, Two Tailed Test

    Pharmacological inhibition of EZH2 and HDAC SAHA increase FOXO1 expression. (A, B ) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 inhibitor GSK126 for 72 h and harvested for western blot analysis (A) of indicated proteins and RT-qPCR analysis of mRNA expression of FOXO1 (B) . ERK2 was used as a loading control for western blot. GAPDH mRNA expression was utilized as an internal control for RT-qPCR. (C, D) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 inhibitor GSK343 for 72 h and harvested for western blot analysis of indicated proteins ( C ) and RT-qPCR analysis of mRNA expression of FOXO1 (D) . ( E, F ) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 ASOs for 48 h and analyses were performed as in A and B . (G, H) C4-2 and 22Rv1 cells were treated with different concentrations of SAHA for 72 h and harvested for western blot (G) and RT-qPCR (H) analysis. (I, J) ChIP-qPCR analysis with H3K27ac antibody (I) and H3K27me3 antibody (J) in C4-2 and 22Rv1 cells. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Figure Legend Snippet: Pharmacological inhibition of EZH2 and HDAC SAHA increase FOXO1 expression. (A, B ) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 inhibitor GSK126 for 72 h and harvested for western blot analysis (A) of indicated proteins and RT-qPCR analysis of mRNA expression of FOXO1 (B) . ERK2 was used as a loading control for western blot. GAPDH mRNA expression was utilized as an internal control for RT-qPCR. (C, D) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 inhibitor GSK343 for 72 h and harvested for western blot analysis of indicated proteins ( C ) and RT-qPCR analysis of mRNA expression of FOXO1 (D) . ( E, F ) C4-2 and 22Rv1 cells were treated with different concentrations of EZH2 ASOs for 48 h and analyses were performed as in A and B . (G, H) C4-2 and 22Rv1 cells were treated with different concentrations of SAHA for 72 h and harvested for western blot (G) and RT-qPCR (H) analysis. (I, J) ChIP-qPCR analysis with H3K27ac antibody (I) and H3K27me3 antibody (J) in C4-2 and 22Rv1 cells. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.

    Techniques Used: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test

    Docetaxel overcomes EZH2 inhibitor resistance in PTEN-mutated cancer cells in culture. (A, B) PTEN-positive 22Rv1 (A) and PTEN-negative C4-2 (B) cells were treated with different concentrations of GSK126 followed by MTS assay at different time points. (C) 22Rv1 and C4-2 cells were treated with different concentrations of GSK126 for 72 h and harvested for western blot analysis with the indicated antibodies. (D) C4-2 cells were treated with vehicle (DMSO) and GSK126 (10 μM) for 72 h followed by further treatment with or without DTX (2 nM) for 30 min prior to IFC. Cell membrane was stained with anti-E-cadherin; the nucleus was counterstained with DAPI. FNS stands for FOXO1 nuclear staining. Scale bar: 25 µm. (E) A hypothetical model deciphers repression of FOXO1 mRNA transcription by EZH2 and regulation of cellular localization of FOXO1 protein by taxane and the PI3K/PTEN/AKT pathway. “P” in a small red circle indicates phosphorylation. (F) C4-2 cells were treated with or without 10 μM of GS126 for 72 h and/or 2 nM of DTX for 30 min prior to fractionation assay and western blot analysis with indicated antibodies. Histone H3 and β-Tubulin were used as nuclear and cytosolic protein marker, respectively. (G - I) C4-2 cells were infected with lentivirus expressing nonspecific shRNAs (shNS) or FOXO1-specific shRNAs and selected with puromycin for stable cell lines. Cells were treated with or without 10 μM of GSK126 and/or 2 nM of DTX for 72 h and harvested for western blot analysis with indicated antibodies (G) , MTS assay (H) , and FACS analysis of percentage of sub G1 cells (I) . Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Figure Legend Snippet: Docetaxel overcomes EZH2 inhibitor resistance in PTEN-mutated cancer cells in culture. (A, B) PTEN-positive 22Rv1 (A) and PTEN-negative C4-2 (B) cells were treated with different concentrations of GSK126 followed by MTS assay at different time points. (C) 22Rv1 and C4-2 cells were treated with different concentrations of GSK126 for 72 h and harvested for western blot analysis with the indicated antibodies. (D) C4-2 cells were treated with vehicle (DMSO) and GSK126 (10 μM) for 72 h followed by further treatment with or without DTX (2 nM) for 30 min prior to IFC. Cell membrane was stained with anti-E-cadherin; the nucleus was counterstained with DAPI. FNS stands for FOXO1 nuclear staining. Scale bar: 25 µm. (E) A hypothetical model deciphers repression of FOXO1 mRNA transcription by EZH2 and regulation of cellular localization of FOXO1 protein by taxane and the PI3K/PTEN/AKT pathway. “P” in a small red circle indicates phosphorylation. (F) C4-2 cells were treated with or without 10 μM of GS126 for 72 h and/or 2 nM of DTX for 30 min prior to fractionation assay and western blot analysis with indicated antibodies. Histone H3 and β-Tubulin were used as nuclear and cytosolic protein marker, respectively. (G - I) C4-2 cells were infected with lentivirus expressing nonspecific shRNAs (shNS) or FOXO1-specific shRNAs and selected with puromycin for stable cell lines. Cells were treated with or without 10 μM of GSK126 and/or 2 nM of DTX for 72 h and harvested for western blot analysis with indicated antibodies (G) , MTS assay (H) , and FACS analysis of percentage of sub G1 cells (I) . Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.

    Techniques Used: MTS Assay, Western Blot, Staining, Fractionation, Marker, Infection, Expressing, Stable Transfection, Two Tailed Test

    Docetaxel overcomes EZH2 inhibitor resistance in PTEN-mutated tumors in mice. (A) C4-2 cells were infected with lentivirus expressing non-specific shRNA (shNS) or a pool of FOXO1-specific shRNAs and selected with puromycin for stable cell lines. Cells were treated with 5 μM of control ASO or EZH2-specific ASO and/or 2 nM of DTX and MTS assay was performed at different time points. (B, C) C4-2 cells were infected with lentivirus expressing shNS or a pool of FOXO1-specific shRNAs as indicated and selected with puromycin. The stable cells (5×10 6 /group) were injected subcutaneously into the right flank of NSG mice. When tumors reached the size of ~100 mm 3 , mice were treated with vehicle (V) (0.9% saline) plus 50 mg/kg of control ASO, vehicle plus 50 mg/kg of EZH2 ASO, 5 mg/kg of DTX plus 50 mg/kg of control ASO or 50 mg/kg of EZH2 ASO plus 5 mg/kg of DTX by i.p. injection twice a week (the 1 st and 4 th day of week). The tumor volume at each time point was documented (B) and tumors in each group were harvested and photographed at day 24 (C) . (D) Western blot analysis of protein expression in xenografts harvested from mice. ERK2 was used as a loading control. The cPARP stands for cleaved PARP. (E, F) H&E and IHC analysis of expression of Ki-67 and cleaved Caspase-3 in xenograft sections from mice with indicated treatment. Representative images are shown in (E) and quantification of Ki67 and cleaved Caspase-3 positive cells from the tissue sections (n = 6) are shown in (F) . The number of positive cells from at least five fields were counted and analyzed. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.
    Figure Legend Snippet: Docetaxel overcomes EZH2 inhibitor resistance in PTEN-mutated tumors in mice. (A) C4-2 cells were infected with lentivirus expressing non-specific shRNA (shNS) or a pool of FOXO1-specific shRNAs and selected with puromycin for stable cell lines. Cells were treated with 5 μM of control ASO or EZH2-specific ASO and/or 2 nM of DTX and MTS assay was performed at different time points. (B, C) C4-2 cells were infected with lentivirus expressing shNS or a pool of FOXO1-specific shRNAs as indicated and selected with puromycin. The stable cells (5×10 6 /group) were injected subcutaneously into the right flank of NSG mice. When tumors reached the size of ~100 mm 3 , mice were treated with vehicle (V) (0.9% saline) plus 50 mg/kg of control ASO, vehicle plus 50 mg/kg of EZH2 ASO, 5 mg/kg of DTX plus 50 mg/kg of control ASO or 50 mg/kg of EZH2 ASO plus 5 mg/kg of DTX by i.p. injection twice a week (the 1 st and 4 th day of week). The tumor volume at each time point was documented (B) and tumors in each group were harvested and photographed at day 24 (C) . (D) Western blot analysis of protein expression in xenografts harvested from mice. ERK2 was used as a loading control. The cPARP stands for cleaved PARP. (E, F) H&E and IHC analysis of expression of Ki-67 and cleaved Caspase-3 in xenograft sections from mice with indicated treatment. Representative images are shown in (E) and quantification of Ki67 and cleaved Caspase-3 positive cells from the tissue sections (n = 6) are shown in (F) . The number of positive cells from at least five fields were counted and analyzed. Data are shown as means ± SEM. The P value was performed by the unpaired two-tailed Student's t-test. * P <0.05; ** P <0.01; *** P <0.001; n.s., no significance.

    Techniques Used: Infection, Expressing, shRNA, Stable Transfection, MTS Assay, Injection, Western Blot, Two Tailed Test

    ezh2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2
    Euchromatic histone methyltransferases (EHMTs) as inversely regulated genes with CXCL10 level in high-risk neuroblastomas and its association with MYCN amplification. (A) Genes correlating with CXCL10 expression in INSS 4 NBs ranked by increasing Pearson correlation coefficients. Epigenetic modifiers from Xu et al are highlighted. Red: positively correlated; blue: negatively correlated. (B) As in (A) but showing genes correlating with MYCN expression. (C) Boxplots showing EHMT2 and EHMT1 expression (RNA-seq, log2) in INSS 4 NBs by MYCN status. (D) Boxplots showing EHMT2 and EHMT1 expression (3′mRNA-seq, log2) in human NB cell lines by MYCN status. (E) Western blots for MYCN, EHMT1, EHMT2, EZH1, <t>EZH2</t> and β-actin in si MYCN or non-targeting non-targeting siRNA (siNT)-treated SK-N-BE and IMR-32 cells. Representative blots of biological replicates (n=4). (F, G) Quantification of experiment described in (E) from biological replicates (n=4). Error bars; SD. (H) and (I) MYCN-binding peaks in the genomic regions of EHMT2 (upper panels) and EZH2 (lower panel) by ChIP-seq. Cell lines and data set as indicated. (J) and (K) EHMT2 and EHMT1 expression in NB cells versus other cancer cells from the CCLE database. Statistics: two-sided unpaired t-tests (C, D, J, K). Two-sided unpaired t-test with logarithmic values (F, G). Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. *p<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. NB, neuroblastoma.
    Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma"

    Article Title: Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2020-001335

    Euchromatic histone methyltransferases (EHMTs) as inversely regulated genes with CXCL10 level in high-risk neuroblastomas and its association with MYCN amplification. (A) Genes correlating with CXCL10 expression in INSS 4 NBs ranked by increasing Pearson correlation coefficients. Epigenetic modifiers from Xu et al are highlighted. Red: positively correlated; blue: negatively correlated. (B) As in (A) but showing genes correlating with MYCN expression. (C) Boxplots showing EHMT2 and EHMT1 expression (RNA-seq, log2) in INSS 4 NBs by MYCN status. (D) Boxplots showing EHMT2 and EHMT1 expression (3′mRNA-seq, log2) in human NB cell lines by MYCN status. (E) Western blots for MYCN, EHMT1, EHMT2, EZH1, EZH2 and β-actin in si MYCN or non-targeting non-targeting siRNA (siNT)-treated SK-N-BE and IMR-32 cells. Representative blots of biological replicates (n=4). (F, G) Quantification of experiment described in (E) from biological replicates (n=4). Error bars; SD. (H) and (I) MYCN-binding peaks in the genomic regions of EHMT2 (upper panels) and EZH2 (lower panel) by ChIP-seq. Cell lines and data set as indicated. (J) and (K) EHMT2 and EHMT1 expression in NB cells versus other cancer cells from the CCLE database. Statistics: two-sided unpaired t-tests (C, D, J, K). Two-sided unpaired t-test with logarithmic values (F, G). Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. *p<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. NB, neuroblastoma.
    Figure Legend Snippet: Euchromatic histone methyltransferases (EHMTs) as inversely regulated genes with CXCL10 level in high-risk neuroblastomas and its association with MYCN amplification. (A) Genes correlating with CXCL10 expression in INSS 4 NBs ranked by increasing Pearson correlation coefficients. Epigenetic modifiers from Xu et al are highlighted. Red: positively correlated; blue: negatively correlated. (B) As in (A) but showing genes correlating with MYCN expression. (C) Boxplots showing EHMT2 and EHMT1 expression (RNA-seq, log2) in INSS 4 NBs by MYCN status. (D) Boxplots showing EHMT2 and EHMT1 expression (3′mRNA-seq, log2) in human NB cell lines by MYCN status. (E) Western blots for MYCN, EHMT1, EHMT2, EZH1, EZH2 and β-actin in si MYCN or non-targeting non-targeting siRNA (siNT)-treated SK-N-BE and IMR-32 cells. Representative blots of biological replicates (n=4). (F, G) Quantification of experiment described in (E) from biological replicates (n=4). Error bars; SD. (H) and (I) MYCN-binding peaks in the genomic regions of EHMT2 (upper panels) and EZH2 (lower panel) by ChIP-seq. Cell lines and data set as indicated. (J) and (K) EHMT2 and EHMT1 expression in NB cells versus other cancer cells from the CCLE database. Statistics: two-sided unpaired t-tests (C, D, J, K). Two-sided unpaired t-test with logarithmic values (F, G). Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. *p<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. NB, neuroblastoma.

    Techniques Used: Amplification, Expressing, RNA Sequencing Assay, Western Blot, Binding Assay, ChIP-sequencing

    Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition restores robust transcriptional responses to IFN-γ and CXCL10 chemokine production in MYCN -amplified human neuroblastoma cells. (A) Western blots H3K27me3 and β-actin in SK-N-BE and IMR-32 cells treated with different PRC2 and EZH2 inhibitors (all 3 µM) for 96 hours. Representative blots of n=3. (B) Western blots for H3K27me3, H3K9me2 and β-actin in SK-N-BE and IMR-32 cells treated with EHMT inhibitor UNC-0638 (2 µM), EZH2 inhibitor EPZ011989 (3 µM) or the combination of both drugs for 96 hours. Representative blots of n=3. (C) CXCL10 mRNA expression assessed by qRT-PCR in SK-N-BE NB cells treated as indicated. Results from biological replicates (n=3). (D) Heatmap visualizing the transcriptional response to IFN-γ in SK-N-BE and (E) IMR-32 cells treated with vehicle or UNC-0638, EPZ011989 or both for 7 days. IFN-γ (250 U/mL) was added for the last 24 hours. Experiments performed in biological replicates (n=3). (F, G) ELISA for CXCL10 protein level (pg/mL) in supernatants from SK-N-BE (F) and IMR-32 NB (G) cells treated as described in (C) and (D). Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.
    Figure Legend Snippet: Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition restores robust transcriptional responses to IFN-γ and CXCL10 chemokine production in MYCN -amplified human neuroblastoma cells. (A) Western blots H3K27me3 and β-actin in SK-N-BE and IMR-32 cells treated with different PRC2 and EZH2 inhibitors (all 3 µM) for 96 hours. Representative blots of n=3. (B) Western blots for H3K27me3, H3K9me2 and β-actin in SK-N-BE and IMR-32 cells treated with EHMT inhibitor UNC-0638 (2 µM), EZH2 inhibitor EPZ011989 (3 µM) or the combination of both drugs for 96 hours. Representative blots of n=3. (C) CXCL10 mRNA expression assessed by qRT-PCR in SK-N-BE NB cells treated as indicated. Results from biological replicates (n=3). (D) Heatmap visualizing the transcriptional response to IFN-γ in SK-N-BE and (E) IMR-32 cells treated with vehicle or UNC-0638, EPZ011989 or both for 7 days. IFN-γ (250 U/mL) was added for the last 24 hours. Experiments performed in biological replicates (n=3). (F, G) ELISA for CXCL10 protein level (pg/mL) in supernatants from SK-N-BE (F) and IMR-32 NB (G) cells treated as described in (C) and (D). Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.

    Techniques Used: Inhibition, Amplification, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Loss of H3K9me2 and H3K27me3 repressive histone marks at CXCL9 and CXCL10 genomic loci on euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibitor treatment of human neuroblastoma cells. Plotted histone ChiP-seq tracks obtained GSE138314 from (A) SK-N-BE and (B) NB1643 MYCN -amplified cells showing the genomic region of CXCL9 , CXCL10 and CXCL11 chemokines genes with neighboring SDAD1 gene on human chromosome 4. (C) As (B), but showing MYCN-binding ChIP-seq track and input obtained from GSE138295. (D) SDAD1 expression (3′mRNA-seq, log2, from PRJEB20874) in SK-N-BE cells transfected with non-targeting siRNA (siNT) and MYCN siRNAs. (E) UCSC genome browser plot showing the genomic regions of SDAD1 , CXCL9 , CXCL10 and CXCL11 with ENCODE ChIP-seq tracks. Strategy of PCR primer pair positioning for tiling ChIP-qPCR in regulatory region of respective genes. Results from biological replicates (n=3) from tiling ChIP-qPCR for (F) H3K27me3 and (G) H3K9me2 represented as percentage input. SK-N-BE cells were treated with indicated inhibitors for 6 days prior to harvesting chromatin. Numbers on x-axis represent primer pairs as described in (E). (H) Scatter plot comparing and correlating baseline level of H3K27me3 and H3K9me2 as (% input) between neighboring SDAD1 and CXCL9 genomic regions based on results from (F) and (G) in untreated SK-N-BE cells. Statistically significant differences between groups ( SDAD1 and CXCL9 ) are indicated. (I) Same analysis as in (H), but for CXCL10 . Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-tests with logarithms of percentage input values; p values corrected for multiple testing with Benjamini and Hochberg method (FDR) in (E) and (G). Error bars: mean±SEM (F, G). Boxplots: boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median.
    Figure Legend Snippet: Loss of H3K9me2 and H3K27me3 repressive histone marks at CXCL9 and CXCL10 genomic loci on euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibitor treatment of human neuroblastoma cells. Plotted histone ChiP-seq tracks obtained GSE138314 from (A) SK-N-BE and (B) NB1643 MYCN -amplified cells showing the genomic region of CXCL9 , CXCL10 and CXCL11 chemokines genes with neighboring SDAD1 gene on human chromosome 4. (C) As (B), but showing MYCN-binding ChIP-seq track and input obtained from GSE138295. (D) SDAD1 expression (3′mRNA-seq, log2, from PRJEB20874) in SK-N-BE cells transfected with non-targeting siRNA (siNT) and MYCN siRNAs. (E) UCSC genome browser plot showing the genomic regions of SDAD1 , CXCL9 , CXCL10 and CXCL11 with ENCODE ChIP-seq tracks. Strategy of PCR primer pair positioning for tiling ChIP-qPCR in regulatory region of respective genes. Results from biological replicates (n=3) from tiling ChIP-qPCR for (F) H3K27me3 and (G) H3K9me2 represented as percentage input. SK-N-BE cells were treated with indicated inhibitors for 6 days prior to harvesting chromatin. Numbers on x-axis represent primer pairs as described in (E). (H) Scatter plot comparing and correlating baseline level of H3K27me3 and H3K9me2 as (% input) between neighboring SDAD1 and CXCL9 genomic regions based on results from (F) and (G) in untreated SK-N-BE cells. Statistically significant differences between groups ( SDAD1 and CXCL9 ) are indicated. (I) Same analysis as in (H), but for CXCL10 . Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-tests with logarithms of percentage input values; p values corrected for multiple testing with Benjamini and Hochberg method (FDR) in (E) and (G). Error bars: mean±SEM (F, G). Boxplots: boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median.

    Techniques Used: ChIP-sequencing, Amplification, Binding Assay, Expressing, Transfection

    High-risk neuroblastomas with high euchromatic histone-lysine methyltransferase (EHMT) and EZH2 activity are characterized by MYCN amplification and a T-cell infiltration-poor tumor microenvironment. (A) Outline of bioinformatic strategy. (B, C, D) Generation of drug response signatures from overlap of differentially expressed genes in SK-N-BE and IMR-32 cells treated with UNC-0638 (B), EPZ011989 (C) or both drugs (D). (E) Exemplary visualization of calculation of EHMT activity scores for high-risk NB samples. (F, G) Exemplary heatmap visualization of expression of UNC-0638 drug response genes in high-risk NB samples ranked by increasing EHMT activity score. (H–J) Heatmaps visualizing immune contexture marker genes (eg, CD8A , CXCL10 ), EHMT2/1 , EZH2/1 and MYCN in high-risk NB samples ranked by increasing activity scores of EHMT (H), EZH2 (I) and EHMT+EZH2 (J). Pearson correlation coefficients are indicated besides the names of the transcripts. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided t-test for Pearson product moment correlation coefficient. NB, neuroblastoma.
    Figure Legend Snippet: High-risk neuroblastomas with high euchromatic histone-lysine methyltransferase (EHMT) and EZH2 activity are characterized by MYCN amplification and a T-cell infiltration-poor tumor microenvironment. (A) Outline of bioinformatic strategy. (B, C, D) Generation of drug response signatures from overlap of differentially expressed genes in SK-N-BE and IMR-32 cells treated with UNC-0638 (B), EPZ011989 (C) or both drugs (D). (E) Exemplary visualization of calculation of EHMT activity scores for high-risk NB samples. (F, G) Exemplary heatmap visualization of expression of UNC-0638 drug response genes in high-risk NB samples ranked by increasing EHMT activity score. (H–J) Heatmaps visualizing immune contexture marker genes (eg, CD8A , CXCL10 ), EHMT2/1 , EZH2/1 and MYCN in high-risk NB samples ranked by increasing activity scores of EHMT (H), EZH2 (I) and EHMT+EZH2 (J). Pearson correlation coefficients are indicated besides the names of the transcripts. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided t-test for Pearson product moment correlation coefficient. NB, neuroblastoma.

    Techniques Used: Activity Assay, Amplification, Expressing, Marker

    Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition amplifies chemokine and MHC I expression by neuroblastomas instigated by low frequency of activated IFN-γ producing T cells. (A) Outline of experimental strategy. (B) Gating strategy and flow cytometric detection of activated CD69 + IFN-γ + human T cells after treatment with anti-CD3/CD28. (C) Quantification of frequency of CD69 + IFN-γ + T cells from three independent donors. (D–G) CXCL9 and CXCL10 mRNA expression by qRT-PCR in SK-N-BE (D, E) and SH-SY5Y (F, G) cells co-cultured and treated as indicated. (H, I) Flow cytometric analysis of MHC I expression on SK-N-BE and SH-SY5Y cells co-cultured and treated as indicated. (J) Model summarizing our findings. Statistics: two-way ANOVA with multiple comparison (C, H). Two-sided unpaired t-test with logarithmic values (D–G). Horizontal line represents median. *P<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. IFN, interferon.
    Figure Legend Snippet: Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition amplifies chemokine and MHC I expression by neuroblastomas instigated by low frequency of activated IFN-γ producing T cells. (A) Outline of experimental strategy. (B) Gating strategy and flow cytometric detection of activated CD69 + IFN-γ + human T cells after treatment with anti-CD3/CD28. (C) Quantification of frequency of CD69 + IFN-γ + T cells from three independent donors. (D–G) CXCL9 and CXCL10 mRNA expression by qRT-PCR in SK-N-BE (D, E) and SH-SY5Y (F, G) cells co-cultured and treated as indicated. (H, I) Flow cytometric analysis of MHC I expression on SK-N-BE and SH-SY5Y cells co-cultured and treated as indicated. (J) Model summarizing our findings. Statistics: two-way ANOVA with multiple comparison (C, H). Two-sided unpaired t-test with logarithmic values (D–G). Horizontal line represents median. *P<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. IFN, interferon.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Cell Culture

    ezh2 d2c9  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2 d2c9
    <t>EZH2</t> was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).
    Ezh2 D2c9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 d2c9/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 d2c9 - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC"

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    Journal: Journal of Cancer

    doi: 10.7150/jca.73291

    EZH2 was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).
    Figure Legend Snippet: EZH2 was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).

    Techniques Used: Expressing

    EZH2 inhibition induces dsRNA expression. (A) EZH2 inhibition induces the mRNA expression of IFNs and ISGs tested by real time qPCR. (B) EZH2 inhibition induces randomly selected ERVs expression tested by real time qPCR. (C) GSEA analysis reveals that the gene sets response to dsRNA was upregulated in GSK126 treated A549 cells vs. control (FDR<0.05). (D-K) The flow cytometry analysis reveals that inhibition of EZH2 induces the expression of dsRNA in NSCLC cell lines A549(D, E), H1299(F, G), H520(H, I), SKMES1(J, K). IFN-γ as a positive control. (*** indicate p<0.001) . (L) Knockdown of EZH2 on protein level was assessed by immunoblotting. (M) The representative IFNs and ISGs mRNA were analyzed by real-time qPCR in EZH2 knockdown A549 cells. (N) The randomly selected ERVs expression was assessed in EZH2 knockdown A549 cells.(O-R) The dsRNA level in EZH2 knockdown A549 cells was tested by flow cytometry (O, P) and immunofluorescence (Q, R). Quantification of dsRNA MFI was followed.
    Figure Legend Snippet: EZH2 inhibition induces dsRNA expression. (A) EZH2 inhibition induces the mRNA expression of IFNs and ISGs tested by real time qPCR. (B) EZH2 inhibition induces randomly selected ERVs expression tested by real time qPCR. (C) GSEA analysis reveals that the gene sets response to dsRNA was upregulated in GSK126 treated A549 cells vs. control (FDR<0.05). (D-K) The flow cytometry analysis reveals that inhibition of EZH2 induces the expression of dsRNA in NSCLC cell lines A549(D, E), H1299(F, G), H520(H, I), SKMES1(J, K). IFN-γ as a positive control. (*** indicate p<0.001) . (L) Knockdown of EZH2 on protein level was assessed by immunoblotting. (M) The representative IFNs and ISGs mRNA were analyzed by real-time qPCR in EZH2 knockdown A549 cells. (N) The randomly selected ERVs expression was assessed in EZH2 knockdown A549 cells.(O-R) The dsRNA level in EZH2 knockdown A549 cells was tested by flow cytometry (O, P) and immunofluorescence (Q, R). Quantification of dsRNA MFI was followed.

    Techniques Used: Inhibition, Expressing, Flow Cytometry, Positive Control, Western Blot, Immunofluorescence

    (A to F) EZH2 inhibition causes dsRNA sensor upregulation and triggers IFNs activation. (G to R) EZH2 abrogation inhibits tumor growth both in vitro and in vivo . (A) Heatmaps for differential expression of gene response to dsRNA (FDR<0.05).(B) Pattern recognition receptors, TLR3, RIG-I, and MDA5, were analyzed by real-time qPCR on mRNA level. (C) TLR3 and MDA5 protein levels were tested by immunoblotting in EZH2 knockdown A549 cells. (D, E) Real-time qPCR analysis of IFNβ (D) and IL-28β (E) upon knockdown with targeting EZH2 alone or combining with pattern recognition receptors.(F) Immunoblotting analysis of ISG15 expression when knockdown with indicated shRNA in A549 cells.(G, H) EZH2 knockdown induces dsRNA expression in LLC cells (G), and dsRNA MFI followed (H). (I, J) Colony formation in NSLCL and LLC cells with EZH2 scramble or knockdown was quantified.(K, L) Colony formation in LLC with scramble, EZH2 knockdown, or EZH2 and MDA5 double knockdown was quantified. (M) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD LLC cells in C57BL6 mice. (N) Representative images of tumors in C57BL6 mice from the scramble group and the EZH2 KD group. (O, P) Representative lung metastasis images (O) and quantification (P) of immunocompetent mice receiving scramble or EZH2 KD B16 cells intravenously. (Q) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD, or EZH2 and MDA5 double KD of LLC cells in C57BL6 mice. (R) Tumor growth of immunocompetent (WT) or immunodeficient (Rag2 -/- ) mice injected with scramble or EZH2 knockdown LLC cells. One-way ANOVA or two-tailed Student's t-test was performed for statistical analysis; *P < 0.05, **P < 0.01.
    Figure Legend Snippet: (A to F) EZH2 inhibition causes dsRNA sensor upregulation and triggers IFNs activation. (G to R) EZH2 abrogation inhibits tumor growth both in vitro and in vivo . (A) Heatmaps for differential expression of gene response to dsRNA (FDR<0.05).(B) Pattern recognition receptors, TLR3, RIG-I, and MDA5, were analyzed by real-time qPCR on mRNA level. (C) TLR3 and MDA5 protein levels were tested by immunoblotting in EZH2 knockdown A549 cells. (D, E) Real-time qPCR analysis of IFNβ (D) and IL-28β (E) upon knockdown with targeting EZH2 alone or combining with pattern recognition receptors.(F) Immunoblotting analysis of ISG15 expression when knockdown with indicated shRNA in A549 cells.(G, H) EZH2 knockdown induces dsRNA expression in LLC cells (G), and dsRNA MFI followed (H). (I, J) Colony formation in NSLCL and LLC cells with EZH2 scramble or knockdown was quantified.(K, L) Colony formation in LLC with scramble, EZH2 knockdown, or EZH2 and MDA5 double knockdown was quantified. (M) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD LLC cells in C57BL6 mice. (N) Representative images of tumors in C57BL6 mice from the scramble group and the EZH2 KD group. (O, P) Representative lung metastasis images (O) and quantification (P) of immunocompetent mice receiving scramble or EZH2 KD B16 cells intravenously. (Q) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD, or EZH2 and MDA5 double KD of LLC cells in C57BL6 mice. (R) Tumor growth of immunocompetent (WT) or immunodeficient (Rag2 -/- ) mice injected with scramble or EZH2 knockdown LLC cells. One-way ANOVA or two-tailed Student's t-test was performed for statistical analysis; *P < 0.05, **P < 0.01.

    Techniques Used: Inhibition, Activation Assay, In Vitro, In Vivo, Expressing, Western Blot, shRNA, Injection, Two Tailed Test

    EZH2 Inhibition enhances lung tumor immunogenicity. (A) Average tumor growth curves of C57BL6 mice inoculated with LLC cells and treated with anti-PD-1 or isotype control. Arrows indicate time points of 100ug/mouse anti-PD-1 injection. (B, C) The tumor infiltration lymphocytes gate strategy (B) and representative dot plot of CD8 + T cells (C) were shown. (D to G) Tumor infiltrating lymphocytes were analyzed by flow cytometry from LLC tumors (scramble n=5, EZH2 KD n=5), the number/gram of CD8 + T (D), CD4 + T (E), MDSC (F), and the CD8 + T/MDSC ratio (G) was shown. (H, I) MHC-I level of LLC tumor isolated from C57BL6 mice was analyzed by flow cytometry, representative dot plot (H), and the MFI was followed. Unpaired t-test was used for statistical analysis. Images are representative of two biological replicates. MFI error bar presents as mean ± SD. *p < 0.05, **p < 0.01, ns, not significant.
    Figure Legend Snippet: EZH2 Inhibition enhances lung tumor immunogenicity. (A) Average tumor growth curves of C57BL6 mice inoculated with LLC cells and treated with anti-PD-1 or isotype control. Arrows indicate time points of 100ug/mouse anti-PD-1 injection. (B, C) The tumor infiltration lymphocytes gate strategy (B) and representative dot plot of CD8 + T cells (C) were shown. (D to G) Tumor infiltrating lymphocytes were analyzed by flow cytometry from LLC tumors (scramble n=5, EZH2 KD n=5), the number/gram of CD8 + T (D), CD4 + T (E), MDSC (F), and the CD8 + T/MDSC ratio (G) was shown. (H, I) MHC-I level of LLC tumor isolated from C57BL6 mice was analyzed by flow cytometry, representative dot plot (H), and the MFI was followed. Unpaired t-test was used for statistical analysis. Images are representative of two biological replicates. MFI error bar presents as mean ± SD. *p < 0.05, **p < 0.01, ns, not significant.

    Techniques Used: Inhibition, Injection, Flow Cytometry, Isolation

    The clinicopathological parameters in NSCLC patients for the OS analysis
    Figure Legend Snippet: The clinicopathological parameters in NSCLC patients for the OS analysis

    Techniques Used: Expressing

    ezh2 antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98

    Structured Review

    Cell Signaling Technology Inc ezh2 antibody
    <t>EZH2</t> could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.
    Ezh2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 antibody/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 antibody - by Bioz Stars, 2023-02
    98/100 stars

    Images

    1) Product Images from "EZH2-mediated epigenetic suppression of lncRNA PCAT18 predicts a poor prognosis and regulates the expression of p16 by interacting with miR-570a-3p in gastric cancer"

    Article Title: EZH2-mediated epigenetic suppression of lncRNA PCAT18 predicts a poor prognosis and regulates the expression of p16 by interacting with miR-570a-3p in gastric cancer

    Journal: Journal of Cancer

    doi: 10.7150/jca.63415

    EZH2 could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.
    Figure Legend Snippet: EZH2 could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.

    Techniques Used: Expressing, Modification, Western Blot

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98
    Cell Signaling Technology Inc ezh2
    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for <t>EZH2,</t> EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc 5246s
    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for <t>EZH2,</t> EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).
    5246s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5246s/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    5246s - by Bioz Stars, 2023-02
    86/100 stars
      Buy from Supplier

    98
    Cell Signaling Technology Inc rabbit monoclonal anti ezh2
    FOXP3 interacts with <t>EZH2</t> in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.
    Rabbit Monoclonal Anti Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal anti ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal anti ezh2 - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    98
    Cell Signaling Technology Inc ezh2 5246 antibody
    Paired primer sequences used in qRT-PCR
    Ezh2 5246 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 5246 antibody/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 5246 antibody - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    98
    Cell Signaling Technology Inc anti ezh2
    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of <t>EZH2,</t> SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.
    Anti Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ezh2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ezh2 - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    98
    Cell Signaling Technology Inc ezh2 d2c9
    <t>EZH2</t> was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).
    Ezh2 D2c9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 d2c9/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 d2c9 - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    98
    Cell Signaling Technology Inc ezh2 antibody
    <t>EZH2</t> could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.
    Ezh2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ezh2 antibody/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ezh2 antibody - by Bioz Stars, 2023-02
    98/100 stars
      Buy from Supplier

    Image Search Results


    Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).

    Journal: Journal of Cancer

    Article Title: Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin

    doi: 10.7150/jca.63234

    Figure Lengend Snippet: Metformin upregulated global level of H3K27ac in vitro . (A) Cells treated with metformin (vehicle, 5 and 10 mM) for 6 days were subjected to western blotting analysis. H3 served as the reference protein for H3K27ac and H3K27me3, and β-actin was used as reference protein for EZH2, EED and SUZ12. (B) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27me3. (C) Tumor tissues of nude mice were sectioned to conduct immunohistochemistry stain for antibody of H3K27ac. (D) IHC scores of H3K27me3 (left) and H3K27ac (right).

    Article Snippet: The primary antibodies were used as following: β-actin (1:2000, 20536-1-AP, Protein tech, China), EZH2 (1:1000, #5246, Cell Signaling Technology, USA), SUZ12 (1:1000, #3737, Cell signaling Technology, USA), EED (1:1000, 16818-1-AP, Protein tech, China), Histone 3 (1:2000, 17168-1-AP Protein tech, China), H3K27me3 (1:1000, #9733, Cell Signaling Technology, USA), and H3K27ac (1:1000, #8173, Cell Signaling Technology, USA).

    Techniques: In Vitro, Western Blot, Immunohistochemistry, Staining

    FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: FOXP3 interacts with EZH2 in murine induced Tregs (iTreg) and freshly isolated PBMC-derived human Tregs. ( A ) Sketch depicts PLA to detect and quantify protein–protein interactions [A] and [B] <30 nm in close proximity or protein modifications by combining ligation of detection probes with rolling-circle amplification. ( B ) Mouse naive CD4 + T cells isolated from spleen differentiated into Tregs (induced) or Th17 cells followed by PLA. Representative confocal PLA images of CD4 + T-cell subsets from 3 independent experiments show endogenous FOXP3–EZH2 protein interaction (red). ( C ) Quantification of PLA + cells from panel B . n = number of cells imaged. *** P < .001. Red horizontal bar shows means ± SEM from 3 independent experiments (1-way analysis of variance + Bonferroni test). ( D ) Whole-cell lysates from activated CD4 + T cells or iTregs in panel B were subjected to immunoprecipitation with anti-FOXP3 and immunoblotted for FOXP3 and EZH2; input shows EZH2 protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( E ) Representative PLA images of PBMC-derived human Tregs (CD4 + CD25 ++ ) from 3 healthy donors showing endogenous FOXP3–EZH2 interaction (magenta) before and after T-cell–receptor activation with antibodies against CD3 and CD28; CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Data are representative of 3 independent experiments. ( F ) Quantification of nuclear PLA signals (number of dots per cell) in images from E . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).

    Techniques: Isolation, Derivative Assay, Ligation, Amplification, Immunoprecipitation, Expressing, Activation Assay

    FOXP3 constitutively interacts with the PRC2 complex. HEK293T cells transfected with plasmids encoding either EZH2 (myc-tagged EZH2), FOXP3 (His-tagged FOXP3), or both for 48 hours were subjected to PLA or co-immunoprecipitation using the indicated primary antibodies. ( A ) Representative PLA images of cells from 3 independent experiments; first 3 rows are negative control experiments and green signals indicate a FOXP3–EZH2 interaction. Scale bar : 20 μm. ( B ) Quantitation of nuclear PLA signals in images from panel A ; n = number of cells imaged. *** P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Whole-cell lysates from HEK293T cells transfected with plasmids encoding His–FOXP3 and myc–DDK–tagged EZH2 were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody, immunoblotted for His–FOXP3 and myc–EZH2 with FOXP3 and myc antibodies. Input shows protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( D ) Cells from panel C were subjected to PLA using His antibody (negative control) or both His and myc antibodies. Red signals indicate FOXP3–EZH2 interaction; data are representative of 3 independent experiments. Scale bar : 20 μm. ( E ) Quantitation from 3 independent experiments of nuclear PLA signals in images from <xref ref-type=Figure 1 D . ∗∗∗ P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test). n = number of cells imaged. ( F and G ) Cell lysates from cell lines transfected with the indicated plasmids (FOXP3 and EZH2 in HEK293T cells or FOXP3 alone in Jurkat T cells) were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody; immunoblotted for His–FOXP3, myc–EZH2, and the other PRC2 subunits SUZ12 and EED with the indicated antibodies. Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole. " width="100%" height="100%">

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: FOXP3 constitutively interacts with the PRC2 complex. HEK293T cells transfected with plasmids encoding either EZH2 (myc-tagged EZH2), FOXP3 (His-tagged FOXP3), or both for 48 hours were subjected to PLA or co-immunoprecipitation using the indicated primary antibodies. ( A ) Representative PLA images of cells from 3 independent experiments; first 3 rows are negative control experiments and green signals indicate a FOXP3–EZH2 interaction. Scale bar : 20 μm. ( B ) Quantitation of nuclear PLA signals in images from panel A ; n = number of cells imaged. *** P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Whole-cell lysates from HEK293T cells transfected with plasmids encoding His–FOXP3 and myc–DDK–tagged EZH2 were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody, immunoblotted for His–FOXP3 and myc–EZH2 with FOXP3 and myc antibodies. Input shows protein expression in whole-cell lysates. Data are representative of 3 independent experiments. ( D ) Cells from panel C were subjected to PLA using His antibody (negative control) or both His and myc antibodies. Red signals indicate FOXP3–EZH2 interaction; data are representative of 3 independent experiments. Scale bar : 20 μm. ( E ) Quantitation from 3 independent experiments of nuclear PLA signals in images from Figure 1 D . ∗∗∗ P < .001. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test). n = number of cells imaged. ( F and G ) Cell lysates from cell lines transfected with the indicated plasmids (FOXP3 and EZH2 in HEK293T cells or FOXP3 alone in Jurkat T cells) were subjected to immunoprecipitation with IgG or anti-FOXP3 antibody; immunoblotted for His–FOXP3, myc–EZH2, and the other PRC2 subunits SUZ12 and EED with the indicated antibodies. Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).

    Techniques: Transfection, Immunoprecipitation, Negative Control, Quantitation Assay, Expressing

    FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P < .0001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( C ) Jurkat cells expressing vector or FOXP3 (WT or mutants) plus luciferase plasmids were treated with DMSO or PMA/ionomycin as in panel B . Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. *** P < .001 (1-way analysis of variance + Bonferroni test). ( D and E ) Whole-cell lysates from HEK293T cells co-expressing myc-tagged EZH2 and His–FOXP3 WT or mutants were subjected to FOXP3 immunoprecipitation, immunoblotted for His–FOXP3 and myc–EZH2 using the indicated antibodies. Red arrows and gray arrows emphasize C232 mutants within FOXP3. Data shown are representative of 3 independent experiments. ( F ) Jurkat cells expressing empty vector or His–FOXP3 (WT or C232 mutants) and indicated luciferase plasmids were treated with 0.4% DMSO or PMA/ionomycin. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates; *** P < .001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( G ) Chromatin from Jurkat cells expressing FOXP3–WT or –C232Δ were incubated with IgG control or anti-H3K27me3 antibody, polymerase chain reaction (PCR) for the IFNγ promoter was performed to assess the presence of H3K27me3 repressive mark. Data shown are representative of 3 independent experiments. ( H ) Jurkat cells overexpressing empty His-vector plasmid (control), His–FOXP3 WT or His–FOXP3 mutants (C232G and C232Δ) were treated with 0.2% DMSO ( top row ) or PMA/ionomycin ( bottom row ) for 12 hours. Cells were permeabilized and then stained with fluorescently conjugated IL2 and His antibodies against intracellular IL2 and His-tagged FOXP3. Dot plot in quadrant 2 (Q2) depicts the frequency, in percentage, of IL2 and His co-expressing cells as measured by flow cytometry. Data shown are representative of 3 independent experiments.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: FOXP3 cysteine 232 to glycine (FOXP3–C232G) mutation implicated in loss of Treg-suppressor function and early onset IBD disrupts EZH2 interaction and its gene co-repressor function. ( A ) Schematic depicts structural domains within 50 kilodalton human FOXP3 (1–431 amino acids), detailing genetic mutations associated with IBD and IPEX syndrome. Structural domains include the following repressor domain (RD), zinc finger (ZnF), LZ, and forkhead (FKH)-DNA binding. Red and black arrows indicate amino acids mutated in IBD and IPEX patients. C232G, cysteine 232 to glycine; L242P, leucine 242 to proline; K250Δ, lysine 250 deletion. ( B ) Jurkat cells transfected with IL2 firefly and renilla luciferase plasmids plus either empty vector or His–FOXP3 plasmids were treated with 0.4% dimethyl sulfoxide (DMSO)–vehicle control or indicated concentrations of EZH2 inhibitor GSK126. Thirty-six hours later, cells were treated with 0.4% DMSO–vehicle control or PMA/ionomycin for 12 hours to activate the IL2 promoter as measured by firefly luciferase expression normalized to the renilla internal control. Bottom panel : Whole-cell lysates from transfected and GSK126-treated cells were immunoblotted for H3K27me3; the same membrane was stripped and reblotted for H3 as control. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. **** P < .0001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( C ) Jurkat cells expressing vector or FOXP3 (WT or mutants) plus luciferase plasmids were treated with DMSO or PMA/ionomycin as in panel B . Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates. *** P < .001 (1-way analysis of variance + Bonferroni test). ( D and E ) Whole-cell lysates from HEK293T cells co-expressing myc-tagged EZH2 and His–FOXP3 WT or mutants were subjected to FOXP3 immunoprecipitation, immunoblotted for His–FOXP3 and myc–EZH2 using the indicated antibodies. Red arrows and gray arrows emphasize C232 mutants within FOXP3. Data shown are representative of 3 independent experiments. ( F ) Jurkat cells expressing empty vector or His–FOXP3 (WT or C232 mutants) and indicated luciferase plasmids were treated with 0.4% DMSO or PMA/ionomycin. Red horizontal bar denotes the mean IL2 firefly/renilla ratio ± SD from 3 replicates; *** P < .001 (1-way analysis of variance + Bonferroni test). Result is representative of 3 independent experiments. ( G ) Chromatin from Jurkat cells expressing FOXP3–WT or –C232Δ were incubated with IgG control or anti-H3K27me3 antibody, polymerase chain reaction (PCR) for the IFNγ promoter was performed to assess the presence of H3K27me3 repressive mark. Data shown are representative of 3 independent experiments. ( H ) Jurkat cells overexpressing empty His-vector plasmid (control), His–FOXP3 WT or His–FOXP3 mutants (C232G and C232Δ) were treated with 0.2% DMSO ( top row ) or PMA/ionomycin ( bottom row ) for 12 hours. Cells were permeabilized and then stained with fluorescently conjugated IL2 and His antibodies against intracellular IL2 and His-tagged FOXP3. Dot plot in quadrant 2 (Q2) depicts the frequency, in percentage, of IL2 and His co-expressing cells as measured by flow cytometry. Data shown are representative of 3 independent experiments.

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).

    Techniques: Mutagenesis, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Immunoprecipitation, Incubation, Polymerase Chain Reaction, Staining, Flow Cytometry

    IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P < .001, ∗ P < .05. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Quantitation of red fluorescent intensities displayed by representative cells shown in panel A . Data are representative of 3 independent experiments. ( D ) Representative confocal microscopic PLA images of intestinal CD4 + T cells from 3 CD patients showing reduced FOXP3–EZH2 complexes (white signals) in comparison with non-CD control cells. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Scale bar : 2–5 μm. Simple endoscopic scores for CD patients were as follows: patient 1, 18; patient 2 (on 30 mg of prednisone), 10; and patient 3 (on 8 mg of budesonide 1 time daily), 8. ( E ) Lamina propria CD4 + T cells from panel D were stained with fluorescently conjugated FOXP3 antibody and subjected to flow cytometric analysis. Histogram overlay compares FOXP3 expression in isolated CD4 + T cells (control vs CD patients 2 and 3). ( F ) Quantitation of nuclear PLA signals/CD4 + T cells from individual CD patients (patients 1, 2, or 3) vs non-CD CD4 + T cells. n = number of cells imaged. Red horizontal bars denote means ± SEM. **** P < .001; NS, 1-way analysis of variance + Bonferroni test. ( G ) Quantitation of nuclear PLA signals/CD4 + T cells from all 3 CD patients vs non-CD control as shown in panel F . n = number of cells imaged. **** P < .0001. Red horizontal bars indicate means ± SEM (Student t test). ( H ) PBMC-derived human Tregs were treated with DMSO, IL6 (50 ng/mL), or IL6 (50 ng/mL) plus 10 μmol/L ruxolitinib (ruxo.) for 2 hours in serum-free media as in panel A , and then permeabilized and stained for FOXP3 or EZH2 with fluorochrome-conjugated antibody or primary antibody, respectively. Dot plots show the frequency, in percentage, of cells expressing FOXP3 ( top row : quadrant 7 [Q7]) or EZH2 ( bottom row : quadrant 3 [Q3]) as measured by flow cytometric analysis. For negative controls, IgG isotype or fluorescently conjugated secondary antibody were used to stain cells. ( I ) Histograms depict FOXP3 or EZH2 expression in cells from panel H . Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; FI, fluorescent intensity; SSC, side scatter.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: IBD-associated IL6-induced membrane-to-nucleus signaling pathway similarly disrupts FOXP3–EZH2 interaction in a manner reversible by JAK1/2 inhibition. ( A ) Confocal microscopic PLA images shows endogenous FOXP3–EZH2 interaction (red signals) in the nucleus of CD4 + CD25 ++ cells (Tregs) before (0.2%–0.4% dimethyl sulfoxide [DMSO]), after IL6 (50 or 100 ng/mL, 2×) or after IL6 and JAK1/2 inhibitor ruxolitinib pretreatment (10 μmol/L). CD4 + CD25 - cells were used as negative controls. Scale bar : 5 μm. Result is representative of 3 independent experiments using freshly isolated PBMC-derived Tregs from 3 different donors. ( B ) Quantitation of nuclear PLA signals in images from panel A (rows 2–7). n = number of cells imaged. *** P < .001, ∗ P < .05. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments. ( C ) Quantitation of red fluorescent intensities displayed by representative cells shown in panel A . Data are representative of 3 independent experiments. ( D ) Representative confocal microscopic PLA images of intestinal CD4 + T cells from 3 CD patients showing reduced FOXP3–EZH2 complexes (white signals) in comparison with non-CD control cells. Dotted white lines denote the plasma membrane as seen on differential interference contrast images. Scale bar : 2–5 μm. Simple endoscopic scores for CD patients were as follows: patient 1, 18; patient 2 (on 30 mg of prednisone), 10; and patient 3 (on 8 mg of budesonide 1 time daily), 8. ( E ) Lamina propria CD4 + T cells from panel D were stained with fluorescently conjugated FOXP3 antibody and subjected to flow cytometric analysis. Histogram overlay compares FOXP3 expression in isolated CD4 + T cells (control vs CD patients 2 and 3). ( F ) Quantitation of nuclear PLA signals/CD4 + T cells from individual CD patients (patients 1, 2, or 3) vs non-CD CD4 + T cells. n = number of cells imaged. Red horizontal bars denote means ± SEM. **** P < .001; NS, 1-way analysis of variance + Bonferroni test. ( G ) Quantitation of nuclear PLA signals/CD4 + T cells from all 3 CD patients vs non-CD control as shown in panel F . n = number of cells imaged. **** P < .0001. Red horizontal bars indicate means ± SEM (Student t test). ( H ) PBMC-derived human Tregs were treated with DMSO, IL6 (50 ng/mL), or IL6 (50 ng/mL) plus 10 μmol/L ruxolitinib (ruxo.) for 2 hours in serum-free media as in panel A , and then permeabilized and stained for FOXP3 or EZH2 with fluorochrome-conjugated antibody or primary antibody, respectively. Dot plots show the frequency, in percentage, of cells expressing FOXP3 ( top row : quadrant 7 [Q7]) or EZH2 ( bottom row : quadrant 3 [Q3]) as measured by flow cytometric analysis. For negative controls, IgG isotype or fluorescently conjugated secondary antibody were used to stain cells. ( I ) Histograms depict FOXP3 or EZH2 expression in cells from panel H . Data are representative of 3 independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; FI, fluorescent intensity; SSC, side scatter.

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).

    Techniques: Inhibition, Isolation, Derivative Assay, Quantitation Assay, Staining, Expressing

    IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments using cells from PBMC donors. ( E and F ) PBMC-derived human Tregs were treated with IL6 or both IL6 and JAK1/2 inhibitor ruxolitinib for the indicated time points. PLA and confocal microscopic imaging were performed to detect and visualize phosphorylated (p) residues (p-Y705 and p-S727) on STAT3 (red signals) by staining cells with the indicated antibodies. Representative images from 3 independent experiments are shown from 3 different donors. White dotted lines mark the cytoplasm of cells while 4′,6-diamidino-2-phenylindole (DAPI) stains the nuclei blue. Red PLA signals indicate phosphorylated tyrosine residue (Y) 705 on STAT3 in cells stained with both STAT3 and p-STAT3-Y705 antibodies or phosphorylated serine residue (S) 727 on STAT3 in cells stained with both STAT3 and p-STAT3-S727 antibodies. Scale bar : 5 μm. ( G and H ) Quantitation of nuclear PLA signals from images in panels E and F , respectively. n = number of cells imaged. Red horizontal bars denote means ± SEM. ** P < .01 and *** P < .001 (1-way analysis of variance + Bonferroni test) from 3 independent experiments across 3 different donors.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

    doi: 10.1016/j.jcmgh.2018.08.009

    Figure Lengend Snippet: IL6-induced disruption of FOXP3–EZH2 protein interaction correlates with increased STAT3 activation and FOXP3 tyrosine phosphorylation. ( A ) HEK293T cells ectopically expressing plasmids encoding His–FOXP3 and myc–EZH2 were treated with IL6 (50 ng/mL) for the indicated duration under reduced-serum conditions. Whole-cell lysates were subjected to immunoprecipitation with IgG or FOXP3 antibody and immunoblotted for His–FOXP3. The same membrane was stripped and reblotted for myc–EZH2. For input, lysates were immunoblotted for His–FOXP3, myc–EZH2, STAT3, and p-STAT3 (Y705) with their corresponding antibodies. ( B ) Reverse co-immunoprecipitation of experiment in panel A using myc antibody for EZH2. Data are representative of 3 independent experiments. ( C ) Representative confocal PLA images of human CD4 + CD25 ++ cells (Tregs) shows tyrosine phosphorylated FOXP3 (red) in response to IL6 (50 ng/mL) alone or in combination with ruxolitinib (Ruxo.) (10 μmol/L) for the indicated time points. To detect FOXP3 tyrosine phosphorylation, cells were stained with pan p-Tyr antibody and specific FOXP3 antibody as indicated. Data are representative of 3 independent experiments. Scale bar : 2 µ m. ( D ) Quantitation of nuclear PLA signals in images from panel C . n = number of cells imaged. *** P < .001; NS, non-significant P value. Red horizontal bars indicate means ± SEM (1-way analysis of variance + Bonferroni test) from 3 independent experiments using cells from PBMC donors. ( E and F ) PBMC-derived human Tregs were treated with IL6 or both IL6 and JAK1/2 inhibitor ruxolitinib for the indicated time points. PLA and confocal microscopic imaging were performed to detect and visualize phosphorylated (p) residues (p-Y705 and p-S727) on STAT3 (red signals) by staining cells with the indicated antibodies. Representative images from 3 independent experiments are shown from 3 different donors. White dotted lines mark the cytoplasm of cells while 4′,6-diamidino-2-phenylindole (DAPI) stains the nuclei blue. Red PLA signals indicate phosphorylated tyrosine residue (Y) 705 on STAT3 in cells stained with both STAT3 and p-STAT3-Y705 antibodies or phosphorylated serine residue (S) 727 on STAT3 in cells stained with both STAT3 and p-STAT3-S727 antibodies. Scale bar : 5 μm. ( G and H ) Quantitation of nuclear PLA signals from images in panels E and F , respectively. n = number of cells imaged. Red horizontal bars denote means ± SEM. ** P < .01 and *** P < .001 (1-way analysis of variance + Bonferroni test) from 3 independent experiments across 3 different donors.

    Article Snippet: Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).

    Techniques: Activation Assay, Expressing, Immunoprecipitation, Staining, Quantitation Assay, Derivative Assay, Imaging

    Paired primer sequences used in qRT-PCR

    Journal: Cell Journal (Yakhteh)

    Article Title: lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways

    doi: 10.22074/cellj.2021.7010

    Figure Lengend Snippet: Paired primer sequences used in qRT-PCR

    Article Snippet: EZH2 (5246) antibody was obtained from CST.

    Techniques:

    Long non-coding RNAs (lncRNAs)-plasmacytoma variant translocation 1( PVT1 ) directly interacted with enhancer of zeste homolog 2 (EZH2) in non-small cell lung cancer (NSCLC). A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in the NSCLC and adjacent normal tissues (n=30). B. qRT-PCR and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in NSCLC cell lines compared to the normal lung epithelial cells, BEAS-2B. C. Relationship between lncRNA PVT1 and EZH2 verified in A549 cells by RNA pull-down and western blot assays using a biotin-labeled probe of PVT1 (bio-PVT1). D. RNA pull-down and western blot assays used to verify the PVT1 effect on EZH2 level after PVT1 knockdown by treating with si-lncPVT1 or si-NC in A549 cells. Data are mean ± SD of three independent experiments. *; P<0.05, **; P<0.01, ***; P<0.001 compared to the adjacent group or MRC5 group. si-NC; siRNA negative control, Bio-NC; Biotin-labeled negative control, Pull; Pull-down group, and SD; Standard deviation.

    Journal: Cell Journal (Yakhteh)

    Article Title: lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways

    doi: 10.22074/cellj.2021.7010

    Figure Lengend Snippet: Long non-coding RNAs (lncRNAs)-plasmacytoma variant translocation 1( PVT1 ) directly interacted with enhancer of zeste homolog 2 (EZH2) in non-small cell lung cancer (NSCLC). A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in the NSCLC and adjacent normal tissues (n=30). B. qRT-PCR and western blot analysis, respectively for the mRNA and protein expression levels of EZH2 in NSCLC cell lines compared to the normal lung epithelial cells, BEAS-2B. C. Relationship between lncRNA PVT1 and EZH2 verified in A549 cells by RNA pull-down and western blot assays using a biotin-labeled probe of PVT1 (bio-PVT1). D. RNA pull-down and western blot assays used to verify the PVT1 effect on EZH2 level after PVT1 knockdown by treating with si-lncPVT1 or si-NC in A549 cells. Data are mean ± SD of three independent experiments. *; P<0.05, **; P<0.01, ***; P<0.001 compared to the adjacent group or MRC5 group. si-NC; siRNA negative control, Bio-NC; Biotin-labeled negative control, Pull; Pull-down group, and SD; Standard deviation.

    Article Snippet: EZH2 (5246) antibody was obtained from CST.

    Techniques: Variant Assay, Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Labeling, Negative Control, Standard Deviation

    pcDNA-PVT1 vector for overexpression (PVT1) regulated expression of yes-associated protein 1 (YAP1) through enhancer of zeste homolog 2 (EZH2) -mediated microRNA-497(miR-497) promoter methylation. A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for the expression levels of EZH2 , miR-497 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with si-EZH2 or si-NC. B. Western blot analysis for the protein levels of EZH2 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with EZH2 siRNA for knockdown (si-EZH2) or si-NC. C. ChIP–qPCR of EZH2 occupancy and H3K27me3 binding in the miR-497 promoter in H1299 cells with or without PVT1 overexpression. a, b and c represented respectively three pairs of amplification primers for miR-497 promoter; and d and e represented respectively two pairs of amplification primers for U6 promoter as internal references. D. MSP analysis for the methylation level of miR-497 promoter in A549 and H1299 cells treated with si-EZH2 or 5-Aza-dC. E. qRT-PCR analysis for the expression levels of miR-497 and YAP1 in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. F. Western blot analysis for the protein levels of LAST2 and YAP1 phosphorylation in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. Data are mean ± SD of three independent experiments, *; P<0.05, **; P<0.01 compared between siRNA negative control (si-NC) and si-EZH2 groups or compared between groups as shown with a horizontal line.5-aza2-deoxycytidine;A549 and H1299; Two of human lung carcinoma cell lines, ChIP; Chromatin immunoprecipitation, MSP; Methylation-specific polymerase chain reaction, LAST2; Large tumor suppressor kinase 2 protein, and SD; Standard deviation.

    Journal: Cell Journal (Yakhteh)

    Article Title: lncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways

    doi: 10.22074/cellj.2021.7010

    Figure Lengend Snippet: pcDNA-PVT1 vector for overexpression (PVT1) regulated expression of yes-associated protein 1 (YAP1) through enhancer of zeste homolog 2 (EZH2) -mediated microRNA-497(miR-497) promoter methylation. A. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for the expression levels of EZH2 , miR-497 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with si-EZH2 or si-NC. B. Western blot analysis for the protein levels of EZH2 and YAP1 in A549 and H1299 cells after EZH2 knockdown, treated with EZH2 siRNA for knockdown (si-EZH2) or si-NC. C. ChIP–qPCR of EZH2 occupancy and H3K27me3 binding in the miR-497 promoter in H1299 cells with or without PVT1 overexpression. a, b and c represented respectively three pairs of amplification primers for miR-497 promoter; and d and e represented respectively two pairs of amplification primers for U6 promoter as internal references. D. MSP analysis for the methylation level of miR-497 promoter in A549 and H1299 cells treated with si-EZH2 or 5-Aza-dC. E. qRT-PCR analysis for the expression levels of miR-497 and YAP1 in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. F. Western blot analysis for the protein levels of LAST2 and YAP1 phosphorylation in A549 and H1299 cells treated with 5-Aza-dC or combined with PVT1 overexpression. Data are mean ± SD of three independent experiments, *; P<0.05, **; P<0.01 compared between siRNA negative control (si-NC) and si-EZH2 groups or compared between groups as shown with a horizontal line.5-aza2-deoxycytidine;A549 and H1299; Two of human lung carcinoma cell lines, ChIP; Chromatin immunoprecipitation, MSP; Methylation-specific polymerase chain reaction, LAST2; Large tumor suppressor kinase 2 protein, and SD; Standard deviation.

    Article Snippet: EZH2 (5246) antibody was obtained from CST.

    Techniques: Plasmid Preparation, Over Expression, Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Binding Assay, Amplification, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation

    Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.

    Journal: Journal of Cancer

    Article Title: HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer

    doi: 10.7150/jca.59740

    Figure Lengend Snippet: Epigenetic regulation of HOXA5 occurs at its putative promoter region. (A) Schematic depiction of the HOXA5 locus on human chromosome 7. Boxes represent exons, lines represent introns, and arrows show the direction of transcription. The gray bars represent the amplicon sites used in chromatin immunoprecipitation-PCR (ChIP-PCR). (B) ChIP-PCR analysis of the histone modifications H3K4me3, H3K27me3, and H3K9ac in MCF7 and TAMR cells. (C) Western blotting images showing protein levels of the epigenetic modifiers in MCF7 and TAMR cells. β-Actin was used as an internal control. (D) ChIP-PCR analysis of EZH2, SUZ12, EED, JMJD3, and UTX in MCF7 and TAMR cells. All experiments were performed in triplicate. *** p < 0.001 compared with MCF7 by Student's t -test.

    Article Snippet: Anti-HOXA5 (ab82645; Abcam), anti-EZH2 (#5246S; Cell Signaling, MA, USA), anti-SUZ12 (#3737; Cell Signaling), anti-EED (ab96801; Abcam, Cambridge, UK), UTX (ab36938; Abcam), anti-JMJD3 (ab169197; Abcam), anti-AKT (#2967; Cell Signaling), anti-phospho-AKT (Thr308, #9275; Cell Signaling), anti-phospho-AKT (Ser473, #4508; Cell Signaling), anti-p53 (sc-126; Santa Cruz Biotechnology, CA, USA), anti-p21 Waf1/Cip1 (#2947; Cell Signaling), anti-Caspase-7 (#12827; Cell Signaling), anti-cleaved-Caspase-7 (#8438; Cell Signaling), anti-Caspase-9 (#9508; Cell Signaling), anti-cleaved-Caspase-9 (#52873; Cell Signaling), anti-PARP (#9542; Cell Signaling), anti-cleaved-PARP (#5625; Cell Signaling), anti-E-cadherin (ab40772; Abcam), anti-N-cadherin (ab18203; Abcam), anti-ZEB1(ab181451; Abcam), and anti-β-Actin (ab6276; Abcam) antibodies were used to detect each of the respective proteins.

    Techniques: Amplification, Chromatin Immunoprecipitation, Western Blot

    EZH2 was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).

    Journal: Journal of Cancer

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    doi: 10.7150/jca.73291

    Figure Lengend Snippet: EZH2 was elevated in NSCLC and negatively regulate IFNs and APP genes. (A) Human EZH2 expression levels in NSCLC (dotted frame) and other tumor types were analyzed by TIMER 2.0 , The statistical significance computed by the Wilcoxon test is annotated by the number of stars (* p < 0.05, ** p <0.01, *** p < 0.001). (B, C) The EZH2 mRNA expression was negatively associated with overall survival in NSCLC (B) and LUAD (C). (D, E) EZH2 expression is negatively related to the infiltration of CD8 + T cells in LUAD (D) and LUSC (E). (F, H) GSEA analysis reveals that there is significant upregulation in gene sets response to type I IFN (F, p<0.001) and antigen processing and presentation genes (H, p<0.001) in GSK126 treated A549 cells vs. Control. (G, I) Heatmap for differential expression of type I IFN-related genes (G, FDR <0.05) and antigen processing and presentation related genes (I, FDR <0.05) between control and GSK126 treated A549 cells (gene lists see ).

    Article Snippet: Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

    Techniques: Expressing

    EZH2 inhibition induces dsRNA expression. (A) EZH2 inhibition induces the mRNA expression of IFNs and ISGs tested by real time qPCR. (B) EZH2 inhibition induces randomly selected ERVs expression tested by real time qPCR. (C) GSEA analysis reveals that the gene sets response to dsRNA was upregulated in GSK126 treated A549 cells vs. control (FDR<0.05). (D-K) The flow cytometry analysis reveals that inhibition of EZH2 induces the expression of dsRNA in NSCLC cell lines A549(D, E), H1299(F, G), H520(H, I), SKMES1(J, K). IFN-γ as a positive control. (*** indicate p<0.001) . (L) Knockdown of EZH2 on protein level was assessed by immunoblotting. (M) The representative IFNs and ISGs mRNA were analyzed by real-time qPCR in EZH2 knockdown A549 cells. (N) The randomly selected ERVs expression was assessed in EZH2 knockdown A549 cells.(O-R) The dsRNA level in EZH2 knockdown A549 cells was tested by flow cytometry (O, P) and immunofluorescence (Q, R). Quantification of dsRNA MFI was followed.

    Journal: Journal of Cancer

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    doi: 10.7150/jca.73291

    Figure Lengend Snippet: EZH2 inhibition induces dsRNA expression. (A) EZH2 inhibition induces the mRNA expression of IFNs and ISGs tested by real time qPCR. (B) EZH2 inhibition induces randomly selected ERVs expression tested by real time qPCR. (C) GSEA analysis reveals that the gene sets response to dsRNA was upregulated in GSK126 treated A549 cells vs. control (FDR<0.05). (D-K) The flow cytometry analysis reveals that inhibition of EZH2 induces the expression of dsRNA in NSCLC cell lines A549(D, E), H1299(F, G), H520(H, I), SKMES1(J, K). IFN-γ as a positive control. (*** indicate p<0.001) . (L) Knockdown of EZH2 on protein level was assessed by immunoblotting. (M) The representative IFNs and ISGs mRNA were analyzed by real-time qPCR in EZH2 knockdown A549 cells. (N) The randomly selected ERVs expression was assessed in EZH2 knockdown A549 cells.(O-R) The dsRNA level in EZH2 knockdown A549 cells was tested by flow cytometry (O, P) and immunofluorescence (Q, R). Quantification of dsRNA MFI was followed.

    Article Snippet: Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

    Techniques: Inhibition, Expressing, Flow Cytometry, Positive Control, Western Blot, Immunofluorescence

    (A to F) EZH2 inhibition causes dsRNA sensor upregulation and triggers IFNs activation. (G to R) EZH2 abrogation inhibits tumor growth both in vitro and in vivo . (A) Heatmaps for differential expression of gene response to dsRNA (FDR<0.05).(B) Pattern recognition receptors, TLR3, RIG-I, and MDA5, were analyzed by real-time qPCR on mRNA level. (C) TLR3 and MDA5 protein levels were tested by immunoblotting in EZH2 knockdown A549 cells. (D, E) Real-time qPCR analysis of IFNβ (D) and IL-28β (E) upon knockdown with targeting EZH2 alone or combining with pattern recognition receptors.(F) Immunoblotting analysis of ISG15 expression when knockdown with indicated shRNA in A549 cells.(G, H) EZH2 knockdown induces dsRNA expression in LLC cells (G), and dsRNA MFI followed (H). (I, J) Colony formation in NSLCL and LLC cells with EZH2 scramble or knockdown was quantified.(K, L) Colony formation in LLC with scramble, EZH2 knockdown, or EZH2 and MDA5 double knockdown was quantified. (M) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD LLC cells in C57BL6 mice. (N) Representative images of tumors in C57BL6 mice from the scramble group and the EZH2 KD group. (O, P) Representative lung metastasis images (O) and quantification (P) of immunocompetent mice receiving scramble or EZH2 KD B16 cells intravenously. (Q) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD, or EZH2 and MDA5 double KD of LLC cells in C57BL6 mice. (R) Tumor growth of immunocompetent (WT) or immunodeficient (Rag2 -/- ) mice injected with scramble or EZH2 knockdown LLC cells. One-way ANOVA or two-tailed Student's t-test was performed for statistical analysis; *P < 0.05, **P < 0.01.

    Journal: Journal of Cancer

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    doi: 10.7150/jca.73291

    Figure Lengend Snippet: (A to F) EZH2 inhibition causes dsRNA sensor upregulation and triggers IFNs activation. (G to R) EZH2 abrogation inhibits tumor growth both in vitro and in vivo . (A) Heatmaps for differential expression of gene response to dsRNA (FDR<0.05).(B) Pattern recognition receptors, TLR3, RIG-I, and MDA5, were analyzed by real-time qPCR on mRNA level. (C) TLR3 and MDA5 protein levels were tested by immunoblotting in EZH2 knockdown A549 cells. (D, E) Real-time qPCR analysis of IFNβ (D) and IL-28β (E) upon knockdown with targeting EZH2 alone or combining with pattern recognition receptors.(F) Immunoblotting analysis of ISG15 expression when knockdown with indicated shRNA in A549 cells.(G, H) EZH2 knockdown induces dsRNA expression in LLC cells (G), and dsRNA MFI followed (H). (I, J) Colony formation in NSLCL and LLC cells with EZH2 scramble or knockdown was quantified.(K, L) Colony formation in LLC with scramble, EZH2 knockdown, or EZH2 and MDA5 double knockdown was quantified. (M) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD LLC cells in C57BL6 mice. (N) Representative images of tumors in C57BL6 mice from the scramble group and the EZH2 KD group. (O, P) Representative lung metastasis images (O) and quantification (P) of immunocompetent mice receiving scramble or EZH2 KD B16 cells intravenously. (Q) Average tumor growth curves of subcutaneously inoculated with scramble or EZH2 KD, or EZH2 and MDA5 double KD of LLC cells in C57BL6 mice. (R) Tumor growth of immunocompetent (WT) or immunodeficient (Rag2 -/- ) mice injected with scramble or EZH2 knockdown LLC cells. One-way ANOVA or two-tailed Student's t-test was performed for statistical analysis; *P < 0.05, **P < 0.01.

    Article Snippet: Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

    Techniques: Inhibition, Activation Assay, In Vitro, In Vivo, Expressing, Western Blot, shRNA, Injection, Two Tailed Test

    EZH2 Inhibition enhances lung tumor immunogenicity. (A) Average tumor growth curves of C57BL6 mice inoculated with LLC cells and treated with anti-PD-1 or isotype control. Arrows indicate time points of 100ug/mouse anti-PD-1 injection. (B, C) The tumor infiltration lymphocytes gate strategy (B) and representative dot plot of CD8 + T cells (C) were shown. (D to G) Tumor infiltrating lymphocytes were analyzed by flow cytometry from LLC tumors (scramble n=5, EZH2 KD n=5), the number/gram of CD8 + T (D), CD4 + T (E), MDSC (F), and the CD8 + T/MDSC ratio (G) was shown. (H, I) MHC-I level of LLC tumor isolated from C57BL6 mice was analyzed by flow cytometry, representative dot plot (H), and the MFI was followed. Unpaired t-test was used for statistical analysis. Images are representative of two biological replicates. MFI error bar presents as mean ± SD. *p < 0.05, **p < 0.01, ns, not significant.

    Journal: Journal of Cancer

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    doi: 10.7150/jca.73291

    Figure Lengend Snippet: EZH2 Inhibition enhances lung tumor immunogenicity. (A) Average tumor growth curves of C57BL6 mice inoculated with LLC cells and treated with anti-PD-1 or isotype control. Arrows indicate time points of 100ug/mouse anti-PD-1 injection. (B, C) The tumor infiltration lymphocytes gate strategy (B) and representative dot plot of CD8 + T cells (C) were shown. (D to G) Tumor infiltrating lymphocytes were analyzed by flow cytometry from LLC tumors (scramble n=5, EZH2 KD n=5), the number/gram of CD8 + T (D), CD4 + T (E), MDSC (F), and the CD8 + T/MDSC ratio (G) was shown. (H, I) MHC-I level of LLC tumor isolated from C57BL6 mice was analyzed by flow cytometry, representative dot plot (H), and the MFI was followed. Unpaired t-test was used for statistical analysis. Images are representative of two biological replicates. MFI error bar presents as mean ± SD. *p < 0.05, **p < 0.01, ns, not significant.

    Article Snippet: Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

    Techniques: Inhibition, Injection, Flow Cytometry, Isolation

    The clinicopathological parameters in NSCLC patients for the OS analysis

    Journal: Journal of Cancer

    Article Title: EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC

    doi: 10.7150/jca.73291

    Figure Lengend Snippet: The clinicopathological parameters in NSCLC patients for the OS analysis

    Article Snippet: Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

    Techniques: Expressing

    EZH2 could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.

    Journal: Journal of Cancer

    Article Title: EZH2-mediated epigenetic suppression of lncRNA PCAT18 predicts a poor prognosis and regulates the expression of p16 by interacting with miR-570a-3p in gastric cancer

    doi: 10.7150/jca.63415

    Figure Lengend Snippet: EZH2 could epigenetically repress PCAT18 expression by H3K27me3 modification. A. The expression of EZH2 in TCGA data of GC. B. Western blot analysis and qPCR of the expression levels of EZH2after cell treatment with si-EZH2 both in SGC7901as well as AGS cells. C. The expression of PCAT18 following EZH2's knockdown. D. ChIP-qPCR of H3K27me3 and EZH2 of the promoter region of the PCAT18 locus following siRNA treatment aimed at si-EZH2 or si-NC in SGC7901 cells. *P < 0.05, **P < 0.01.

    Article Snippet: The EZH2 antibody (1:1000) were acquired from Cell Signaling Technology (Cat#: 5246), and the GAPDH served as control (CST, Cat#: 5174).

    Techniques: Expressing, Modification, Western Blot