Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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

Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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

Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Control, ChIP-qPCR, Two Tailed Test

Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: MTS Assay, Western Blot, Membrane, Staining, Phospho-proteomics, Fractionation, Marker, Infection, Expressing, Stable Transfection, Two Tailed Test

Journal: Theranostics

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

doi: 10.7150/thno.34700

Figure Lengend 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.

Article Snippet: Another EZH2 inhibitor GSK343 (HY-13500) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 2. RACGAP1 overexpression promoted neuroendocrine transformation in prostate cancer. (A) Western blot analysis showed that RACGAP1 was upregulated in NEPC-like cells. (B) Immunohistochemical study of different types of prostate cancer tissues showed that RACGAP1 was highly expressed in NEPC. (C) Data for quantified immunohistochemistry in adjacent normal tissues (n=10), tumor tissues (n=10) and NEPC (n=7) of prostate cancer are shown as mean + SD. (D) Enzalutamide (MDV3100) induced the production of RACGAP1, and DHT partially reversed this effect. (E) Western blot analysis showed the protein expression of RACGAP1, CHGA, and SYP in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days. (F) The mRNA level of RACGAP1, NCAM, CHGA, SYP, and NSE in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days were determined by qRT-PCR analysis. (G) RACGAP1 and NE markers (CHGA, NCAM, NSE, and SYP) in C4-2 cells following transient transfection with control (shNC) or RACGAP1 shRNA (sh1, sh2), as detected by qRT-PCR. (H) RACGAP1 and NE markers (CHGA, SYP) in C4- 2B cells following transient transfection with RACGAP1 or an empty vector, as detected by qRT-PCR. (I) Protein

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Over Expression, Transformation Assay, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Plasmid Preparation

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 5. RACGAP1 protein interacts with the EZH2 protein and increases the protein stability of EZH2. (A) Analysis of protein-protein interaction (PPI) information shows that RACGAP1 may directly interact with EZH2. (B) GSEA shows that high RACGAP1 expression was enriched in the EZH2-TARGET pathway. (C) RNA levels of RACGAP1 and EZH2 in NE-like cells treated with RACGAP1 or empty vector (CON), as determined by qRT-PCR. (D) Western blot was performed to show protein expression of RACGAP1 and E2F1 in NE-like cells following control, RACGAP1, RACGAP1 shRNA1 (sh1), or RACGAP1 shRNA1 (sh2) transfection. (E) Total cell lysates of NE-like cells were immunoprecipitated with anti-EZH2 or anti- RACGAP1 antibodies and blotted with corresponding antibodies. (F) Representative immunofluorescence images of RACGAP1 and EZH2 protein localization in PC3 cells. (G) C4-2B-N cells and PC3 cells were transiently transfected with CON or RACGAP1 and supplied with 10 mmol/L cycloheximide (CHX), and then, total cell lysates were collected at 0, 8, 16, and 24 h after treatment. Western blot analysis was used to measure protein levels. (H) Protein expression analysis was used to calculate the half-life of EZH2 protein for C4-2B-N and PC3 cells. (I) Cells transiently transfected with RACGAP1 knockdown were treated with vehicle (DMSO), chloroquine (50 μm), or MG132 (20 μm) for 12 h. Western blotting was used to detect the protein level of RACGAP1. Bar graphs show the statistical analysis of three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Transfection, Immunoprecipitation, Immunofluorescence, Knockdown

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 6. RACGAP1 stabilizes EZH2 protein expression in the ubiquitin-proteasome pathway and affects NED in prostate cancer by regulating EZH2. (A) PC3 and DU145 cells were treated with FLAG-RACGAP1 for 48 h. Total cell lysates were subjected to immunoprecipitation with EZH2 antibody and blotted with an anti- ubiquitin antibody. (B) 293T cells were co-transfected with GFP-EZH2, Myc-ubi, and different doses of FLAG- RACGAP1 (0, 2, 4 µg) for 48 h. Total cell lysates were subjected to immunoprecipitation with a GFP antibody and blotted with an anti-Myc antibody. (C) PC3 cells were transfected for rescue experiments, using PC3 cells with an shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid, and cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid. Western blotting was used to detect proteins using the indicated antibodies. We transfected PC3 and DU145 cell lines for rescue experiments, using cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus (RA) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2, and cells with the RACGAP1 expression lentivirus (RA) and shEZH2. (D) Western blotting was used to detect proteins using the indicated antibodies. (E) Transwell assay for indicated PC3 cells (magnification: 100×). Bar graphs showing the statistical analysis of three independent experiments. ***, p < 0.001, **, p < 0.01, *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Western Blot, Transwell Assay

Journal: eLife

Article Title: SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc-SMPDL3B

doi: 10.7554/eLife.67452

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Ezh2 (Rabbit monoclonal) , Cell Signaling , 5246; RRID: AB_10694683 , CHIP(1:100) WB(1:1000).

Techniques: Generated, CRISPR, Plasmid Preparation, Stable Transfection, Expressing, Transfection, Construct, Clone Assay, shRNA, Mutagenesis, Recombinant, Luciferase, Sequencing, In Vitro, Staining, Isolation, Reverse Transcription, SYBR Green Assay, Real-time Polymerase Chain Reaction, Labeling, Northern Blot, Hybridization, Purification, Knockdown, Reporter Assay, Activity Assay, Membrane, Software

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 1. Expression and function of EZH2 in CRC tissues and cells. (A) Immunohistochemical analysis of EZH2 in normal colorectal mucosa, CRC tissues, and CRC tissues from patients with metastasis (original magnification ×400). (B) Western blot analysis showing almost no expression of EZH2 in a normal human colon epithelial cell line, CRL-1790; however, the expression of EZH2 was significantly increased in all CRC cell lines, especially in highly invasive CRC cells. (C) Transwell Matrigel invasion assays were performed in CRC cells infected with control shRNA or EZH2 shRNA viruses or control or EZH2 overexpression virus. Cells were observed under a light microscope and photographed. Cells were counted from five random microscopic fields (200×) per insert in triplicate. The migrated cell numbers were normalized to that of the control group. Data are shown as mean ± SD from three separate experiments. (D) Wound healing assays were performed in CRC cells after infection with the EZH2 overexpression virus, control virus, control shRNA virus or EZH2 shRNA virus. n = 3, ANOVA,*p < 0.05, compared with CRL-1790 cells; n = 3, ANOVA, ▲p < 0.05, compared with the control shRNA virus; n = 3, ▲▲p < 0.05 (Student’s t test).

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Infection, Control, shRNA, Over Expression, Virus, Light Microscopy

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 2. Functional impact of EZH2 overexpression/knockdown on the metastatic potential of CRC cells in vivo. (A) Assessment of the subcutaneous metastatic capacity of 2.5 × 106 SW1116-GFP-control or SW1116-GFP-EZH2 cells after inoculating nude mice via tail vein injection. Biofluorescence images and summarized data show subcutaneous metastasis and total photon flux for each treatment group (a: mice injected with PBS; b: mice injected with SW1116-GFP-control stable cells; c: mice injected with SW1116-GFP-EZH2 stable cells). n = 15, ANOVA, *p < 0.0001. (B) Assessment of subcutaneous metastatic capacity of 2.5 × 106 HCT116-GFP-control shRNA or HCT116-GFP-shEZH2-1/2 tumour cells by inoculating nude mice via tail vein injection. Biofluorescence images and summarized data show subcutaneous metastasis and total photon flux for each treatment group (d: mice injected with PBS; e: mice injected with HCT116-GFP-control shRNA stable cells; f1: mice injected with HCT116-GFP-shEZH2-1 stable cells; f2: HCT116-GFP-shEZH2-2 stable cells). n = 15, ANOVA, *p < 0.0001. (C, D) Survival curves for mice. n = 15, **p < 0.05 (Student’s t-test), compared with SW1116-GFP-control cells or HCT116-GFP-control shRNA cells. (E) Increased numbers of metastatic CRC cells were detected in the lungs of nude mice at 17 weeks after the injection of the SW1116-GFP-EZH2 group compared with the SW1116-GFP-control group. Fewer metastatic CRC cells were detected in the lungs of nude mice injected with HCT116-GFP-shEZH2-1/2 cells at 17 weeks compared with mice injected with HCT116-GFP control shRNA cells.(F) Survival analysis showed that EZH2-positive tumours have an unfavourable prognosis compared with EZH2-negative tumours in patients (p < 0.05, HR = 0.1973, 95% CI 0.06–0.66).

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Functional Assay, Over Expression, Knockdown, In Vivo, Control, Injection, shRNA

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 3. Role of EZH2 in VDR regulation and effect of VDR on EZH2-induced CRC cell invasion. (A) Real-time PCR data show that infection of EZH2 overexpression virus dramatically increased EZH2 mRNA expression and decreased VDR mRNA expression in SW1116 cells. n = 3, *p < 0.01 (Student’s t-test). (B) Real-time PCR show that EZH2 shRNA virus significantly decreased the mRNA level of the EZH2 gene and increased the mRNA level of the VDR gene in HCT116 CRC cells. n = 3, **p < 0.01 (Student’s t-test). (C)Western blot analysis data show that overexpression of EZH2 significantly decreased the expression of VDR in SW1116 cells. n = 3, *p < 0.01 (Student’s t-test). (D) Knockdown of EZH2 dramatically increased the expression of VDR in HCT116 cells. n = 3, **p < 0.01 (Student’s t-test). (E, F) Transwell Matrigel invasion assays were performed in CRC cells infected with different viruses as indicated. Cells were observed under a light microscope and photographed. Cells were counted from five random microscopic fields (200×) per insert in triplicate. The migrated cell numbers were normalized to that of the control group. Data are shown as mean ± SD from three separate experiments. n = 3, ANOVA, p < 0.01, compared with control; p < 0.05, compared with EZH2 overexpression virus or EZH2 shRNA virus.

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Real-time Polymerase Chain Reaction, Infection, Over Expression, Virus, Expressing, shRNA, Western Blot, Knockdown, Light Microscopy, Control

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 4. The mechanism of EZH2-mediated VDR down-regulation in CRC cells. (A) Knockdown of EZH2 significantly increased the luciferase activity of the VDR gene promoter. n = 3, *p = 0.0005 (Student’s t-test). (B) The band in the gel of the ChIP assay suggests that the H3K27me3 site exists in the VDR gene promoter. Input DNA was used as a positive control; cell lysates incubated with non-relevant rabbit IgG or cell lysate without antibody incubation were used as negative controls. (C) Real-time PCR of ChIP samples showed that knockdown of EZH2 dramatically decreased the trimethylation level of H3K27 in the VDR promoter in HCT116 cells. n = 3, **p = 0.0097 (Student’s t-test). (D) Immunohistochemical analysis of consecutive human CRC tissue sections showed that higher levels of EZH2 nuclear staining were observed in CRC samples than in normal colonic epithelial samples, and the expression of VDR showed the opposite trend (original magnification ×400).

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Knockdown, Luciferase, Activity Assay, Positive Control, Incubation, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 5. Effect of STAT3 on EZH2 and VDR expression in CRC cells. Western blot analysis data show that infection of STAT3 shRNA virus dramatically inhibited the phosphorylation and expression of STAT3. STAT3 down-regulation significantly decreased the expression of EZH2 and increased the expression of VDR in SW1116 (A) and HCT116 cells (B). *p < 0.01 (Student’s t-test), compared with control shRNA virus. Western blot analysis data show that the phosphorylation and expression of STAT3 were successfully increased after introducing STAT3 overexpression virus. EZH2 expression was significantly increased and VDR expression was decreased after the overexpression of STAT3 in SW1116 (C) and HCT116 cells (D). n = 3, **p < 0.01 (Student’s t-test), compared with control virus. Real-time PCR and western blot data show that introducing EZH2 shRNA virus significantly blocked STAT3-mediated VDR down-regulation in CRC cells (E, F). ANOVA, p < 0.05, compared with control virus group; p < 0.05, compared with STAT3 overexpression virus group.

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Infection, shRNA, Virus, Phospho-proteomics, Control, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 6. The mechanism of STAT3-mediated EZH2 up-regulation in CRC cells. (A) STAT3 up-regulation dramatically increased the luciferase activity of the EZH2 gene promoter, suggesting that STAT3 regulates the expression of EZH2 by modulating the transcriptional activity of the EZH2 gene. n = 3, *p < 0.01 (Student’s t-test). (B) STAT3 binding sites in the EZH2 gene promoter. White and black triangles indicate a wild-type or mutant sequence for STAT3 binding sites, respectively. WT = wild type; MT = mutant type of each STAT3 mutation binding site. Mutation of STAT3 binding sites significantly decreased the transcriptional activity of the EZH2 promoter in the luciferase assay. n = 3, *p < 0.01 (Student’s t-test). (C) Bioinformatic analysis of STAT3 transcriptional factor binding sites in part of the EZH2 gene promoter region. Numbers on the left-hand side indicate the locations upstream of the first base of the initial translation site. STAT3 binding sites are highlighted, and the DNA sequence encompassed by two arrows was amplified in the ChIP assay. EZH2 DNA was detected in the chromatin sample immunoprecipitated from HCT116 cells using an antibody against STAT3 (D), suggesting that STAT3 binds to the EZH2 promoter. (E) Real-time PCR of the ChIP samples showed that overexpression of STAT3 dramatically increased the binding efficiency of STAT3 to the EZH2 promoter in CRC cells. ChIP assay real-time PCR was performed using GAPDH as a negative control for STAT3 binding. n = 3, **p < 0.01 (Student’s t-test). (F) Transwell Matrigel invasion assays were performed in CRC cells infected with different viruses as indicated. Cells were observed under a light microscope and photographed. Cells were counted from five random microscopic fields (200×) per insert in triplicate. The migrated cell numbers were normalized to that of the control group. Data are shown as mean ± SD of three separate experiments. n = 3, ANOVA, p < 0.01, compared with control virus plus control shRNA virus; p < 0.05, compared with STAT3 overexpression virus plus control shRNA virus. (G) Immunohistochemical analysis for pSTAT3 and EZH2 in consecutive human CRC tissue sections. EZH2 and pSTAT3 were expressed at higher levels in CRC than in normal colonic epithelial samples, and the expression of VDR showed the opposite trend. There was no significant difference in STAT3 expression in normal colonic epithelial samples or CRC tissues (original magnification ×400).

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Luciferase, Activity Assay, Expressing, Binding Assay, Mutagenesis, Sequencing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Over Expression, Negative Control, Infection, Light Microscopy, Control, Virus, shRNA, Immunohistochemical staining

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 7. Expression levels of pSTAT3, STAT3, EZH2, and VDR in a nude mouse xenograft model of CRC. Immunohistochemical analysis of pSTAT3, STAT3, EZH2, and VDR in xenograft tissue sections. STAT3 was successfully up-regulated or down-regulated after introducing STAT3 shRNA and the STAT3 overexpression virus into nude mouse CRC cell model, respectively (A, B). The level of pSTAT3 and the expression of EZH2 were significantly up-regulated in tumour samples with STAT3 overexpression (C), while the levels of pSTAT3 and EZH2 expression were significantly down-regulated in STAT3-knockdown tumour samples. The expression of VDR displayed an opposite trend (D). (A–D) Original magnification: ×400.

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Immunohistochemical staining, shRNA, Over Expression, Virus, Knockdown

Journal: The Journal of pathology

Article Title: Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

doi: 10.1002/path.4179

Figure Lengend Snippet: Figure 8. Model for the possible mechanism of EZH2-induced invasion in CRC and the regulatory mechanism of EZH2. Activation of STAT3 in CRC cells leads to overexpression of EZH2, which results in decreased expression of VDR and increased metastatic ability in CRC cells.

Article Snippet: The protein–DNA complexes were precipitated using 5 μg of antibodies against trimethyl-histone 3-Lys27 (3meH3K27; #07–449, BD San Jose, CA, USA), STAT3 (#9132, CST), EZH2 (#4905, CST, San Jose, CA, USA) or IgG control (#12–370, Santa Cruz, Dallas, TX, USA).

Techniques: Activation Assay, Over Expression, Expressing

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: The association of EZH2 and JMJD3 expressions with HSC activation and liver fibrosis. ( A ) The expressions of EZH2, JMJD3, α-SMA, H3K27me2/3 and β-Actin as inner control during culture-induced activation of rat primary HSCs (0, 1, 3, 5, 7 days) was analyzed by Western blot. ( B ) In the gene arrays dataset for liver biopsy samples from 124 liver fibrosis patients (GSE84044, ref 29), linear regression was used to analyze the association of hepatic expression of PRC2 subunit genes ( EZH2 , SUZ12 , EED and RBBP4 ) with sequential histological staging of fibrosis, Scheuer score, S , which is divided as S0 (stage 0, n = 43), S1 ( n = 20), S2 ( n = 33), S3 ( n = 18) and S4 ( n = 10). The box plot and fitting curve showed the association trend. The significance of correlation was expressed as r -squared values of regression and P < 0.01 was considered as statistical significance.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Activation Assay, Control, Western Blot, Expressing

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: EZH2 and JMJD3 regulate HSC activation through modulating Tgfβ1 signaling pathway. Rat primary HSCs were cultured and treated with inhibitors of EZH2 (DZNep) or JMJD3 (GSK-J4), or transfected with siRNAs of Ezh2 , or infected with adenovirus recombined with Jmjd3 . RNA-seq was conducted for HSCs treated with DZNep or DMSO as control. ( A ) HSCs growth and morphology were assessed in fifth day after administration, the magnification is 200×. ( B-D ) EZH2, JMJD3, α-SMA and COL1A expressions and H3K27 methylation level were determined by Western blot with β-ACTIN and Histone 3 as inner controls respectively. * P < 0.05. ( E ) Basing on the DEGs associated with DZNep treatment in HSCs, the upstream regulator analysis of IPA identified Tgfβ1 among the most significantly inhibited growth factors or cytokines. The top candidates are shown with overlap P value < 0.01 and | activation Z -score | >2. (F) The heatmap shows transcriptional value log 10 RPKM for genes encoding effectors, activators and inhibitors of TGFβ1 signaling pathway. Bambi in the inhibitor panel was highlighted in red.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Activation Assay, Cell Culture, Transfection, Infection, RNA Sequencing, Control, Methylation, Western Blot

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: Cellular phenotypic effects of EZH2 inhibition with DZNep or GSK126 on HSCs cell cycling, proliferation, senescence and apoptosis. JS1 cells were administrated with DZNep, GSK126 or DMSO and subjected to analyses of cell cycle ( A ), cell growth ( B-C ), senescence ( D ) and apoptosis ( E-F ) with staining and flow cytometry. The transcriptional expression of cell proliferation marker MKI67 gene in rat primary HSC, human LX-2 and mouse JS cells were measured by RT-qPCR (C). Cell senescence was determined by measuring SA-β-Gal activity (D). The apoptotic cells were determined by Annexin-V-PI double staining (E) and FITC-dUTP staining by TUNEL apoptosis assay (F).The yellow frames in panel E present early apoptotic cells. Data were measured from triple independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Inhibition, Staining, Flow Cytometry, Expressing, Marker, Quantitative RT-PCR, Activity Assay, Double Staining, TUNEL Assay, Apoptosis Assay

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: DZNep treatment results in reduced hepatic expressions of fibrotic marker genes in mice with CCl 4 -induced liver fibrosis and prevalent downregulation of ECM component genes in HSCs. Hepatic expressions of H3K27me3, Histone3, EZH2 and β-Actin in CCl 4 mice with DZNep or DMSO treatment were determined by Western blot ( A-B ). Hepatic transcriptions of Ezh2 , α-Sma , Col1a , Mmp2 and β-Actin were determined by RT-qPCR. The pre-ranked gene list for transcriptomic profiling of rat primary HSCs treated with DZNep or DMSO was subjected to GSEA, ECM genes were significantly enriched ( C ). The heatmap shows transcriptional changes of various ECM component genes ( D ). Statistical significance was determined with Student's t -test in independent samples.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Marker, Western Blot, Quantitative RT-PCR

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: HSC-specific Ezh2 silencing in vivo ameliorated liver fibrosis. ( A ) Construction of amiRNA vector for Ezh2 silencing. The amiRNA consisted of miR-30a backbone as structural scaffold and two concatenated pre-miR30-shRNA cassettes embedding valid interfering shRNA (amiR- Ezh2 ) or negative control shRNA (amiR-NC) as cargos. Based on lentiviral microRNA expressional vector, amiRNA was inserted into the cloning site located in EGFP intron, the original CMV promoter was substituted with human GFAP promoter. ( B ) Ezh2 silencing in JS1 cells was achieved by infection of lentiviral-amiR- Ezh2 , expressions of EZH2, COL1A, α-SMA and β-ACTIN were determined by Western blot. ( C ) In CCl 4 mice, lentivirally expressed amiRNA was transduced into the liver through tail-vein injection, therapeutic effects of amiRNA-based HSC-specific Ezh2 silencing was analyzed by measuring collagens deposition with Masson's trichrome staining and α-SMA expression with immunohistochemistry.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: In Vivo, Plasmid Preparation, shRNA, Negative Control, Cloning, Infection, Western Blot, Injection, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: EZH2 and JMJD3 regulate Bambi , Cdkn1a , Gadd45a and Gadd45b. Primary rat HSCs were treated with DZNep or infected with adenovirus- Jmjd 3 or their controls respectively. The effects of DZNep on H3K27me3 ( A ) or Jmjd3 overexpression on H3K27me2/me3 ( B ) enrichments at target genes were determined by ChIP-qPCR. The 5′ endpoints of PCR products are positioned as upstream (-) or downstream (+) from transcription start sites. The effect of DZNep treatment or Jmjd3 overexpression on transcript and protein products of target genes were determined by RT-qPCR ( C-D ) and Western blot ( E-F ) respectively. β-Actin was used as internal control. Hepatic transcriptions of Bambi and Cdkn1a in CCl 4 ( G ) and BDL ( H ) mice were determined by RT-qPCR. * P < 0.05, ** P < 0.01.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Infection, Over Expression, ChIP-qPCR, Quantitative RT-PCR, Western Blot, Control

Journal: Life Science Alliance

Article Title: DEK promotes mammary hyperplasia and is associated with H3K27me3 epigenetic modifications

doi: 10.26508/lsa.202503230

Figure Lengend Snippet: (A) mRNA expression, quantified as FPKM from bulk RNA-seq data, for ERα ( Esr1 ), Erbb2 (Her2), and other genes associated with luminal hormone receptor positive or basal cell populations. n = 2/group same as . Some markers may be higher in Dek-OE compared with controls, but others appear to be unchanged. (B) Western blotting of whole mammary gland tissue lysates from 6–8-wk-old, virgin female mice revealed no differences in ERa or Her2 protein levels between samples from -dox Dek-OE mice and +dox control mice (n = 2–5/group). Densitometry quantification of Western blots is depicted in the graph on the right. (C) Western blotting of whole mammary gland tissue lysates from 8-wk-old, virgin female mice revealed no differences in progesterone receptor (PR-A and PR-B) protein levels between samples from -dox Dek-OE mice and +dox control mice (n = 3/group, P = 0.2). (D) Coomassie and silver stain of all proteins precipitated with GFP-DEK in a GFP-trap protein interaction assay compared with a GFP-His protein negative control performed in HEK293 cells. Samples were subsequently immunoblotted for PRC2 complex proteins . (E) Western blotting demonstrates that treating MCF10A cells with 2 μM EZH2 inhibitor GSK-126 for 48 h blocks H3K27me3 formation.

Article Snippet: DEK (#610948, 1:1,000; BD Biosciences), SUZ12 (#3737, 1:1,000; Cell Signaling Technology), and EZH2 (#21800-1-AP, 1:2,000; Proteintech) primary antibodies were used to probe for co-immunoprecipitation.

Techniques: Expressing, RNA Sequencing, Western Blot, Control, Silver Staining, Protein Interaction Assay, Negative Control

Journal: Life Science Alliance

Article Title: DEK promotes mammary hyperplasia and is associated with H3K27me3 epigenetic modifications

doi: 10.26508/lsa.202503230

Figure Lengend Snippet: (A) UMAP plot depicting Dek expression (red) in single cell RNA-sequencing data from mouse mammary gland previously published by reference . The populations are mammary stem cells, luminal alveolar progenitor cells (LA-pro), luminal hormone receptor positive progenitors (LH-pro), basal cells, mature luminal alveolar cells (L-Alv), and mature luminal hormone receptor positive cells (L-Hor). (B) Gene set enrichment analysis plots for genes co-expressed with Dek in the luminal alveolar progenitor cellular compartment. Plots depict E2F Targets, Chromatin Organization, Chromatin Remodeling, and Regulation of Gene Expresion-Epigenetic ontologies. (C) A list of the top genes co-expressed with Dek in the luminal alveolar progenitor cell population. (A, D) UMAP plots for gene expression of Ezh2 , and proliferation-associated genes Pcna and E2F1 , overlap with the expression of Dek in (A). (C) All three genes are in the list shown in (C). (E) Dek , Ezh2 , and Rbbp7 mRNA expression levels in each mammary cell population are depicted as a violin plot. All three genes are most highly expressed in MaSC, LA-pro, and LH-pro populations.

Article Snippet: DEK (#610948, 1:1,000; BD Biosciences), SUZ12 (#3737, 1:1,000; Cell Signaling Technology), and EZH2 (#21800-1-AP, 1:2,000; Proteintech) primary antibodies were used to probe for co-immunoprecipitation.

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: Life Science Alliance

Article Title: DEK promotes mammary hyperplasia and is associated with H3K27me3 epigenetic modifications

doi: 10.26508/lsa.202503230

Figure Lengend Snippet: (A) Ezh2 gene expression from bulk RNA-Seq of +dox control versus -dox Dek over-expressing mouse mammary glands from MMTV-tTA/Bi-L-Dek mice, as described in (n = 2/group, 60-wk old virgin females). (B) Mammary glands from Dek-OE mice have increased levels of histone H3 trimethylation on residue lysine 27 (H3K27me3) compared with + dox controls, as shown by western blot. Densitometry quantification is graphed to the right. N = 3 mice/group. Graphs are presented as mean ± SEM and statistical significance is determined by an unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001 ns = not significant. (C) H3K27me3 and EZH2 protein levels are increased in Dek-OE mammary epithelium from 8-wk old virgin female mice, compared with controls, as determined by immunohistochemistry. Staining intensity, quantified as optical density using Image J, is graphed to the right with N = 5 mice/group and statistical significance determined using an unpaired t test. (D) H3K27me3 levels positively correlate with DEK expression in human MCF10A cells either over-expressing DEK (left) or with DEK knockdown with shRNA (right) as determined by western blot of whole cell lysates. DEK over-expression was accomplished with a dox-inducible pTRIPZ vector whereas DEK knockdown was accomplished with a pLKO.1 shRNA vector. (E) Immunoblotting of GFP-trap nuclear lysates identify proteins that interact with GFP-DEK. Interacting proteins include key members of the PRC2 complex, EZH2, RBBP4, and EED, as well as endogenous DEK, histone H3, and the known DEK-interacting positive control of CK2. Input lysates, pre-incubation with GFP trap beads, are shown adjacent to the pull-down results. (D, F) DEK interacts with PRC2 complex members EZH2 and SUZ12 as determined by immunoprecipitation using whole cell lysates from MCF10A cells with dox-induced DEK overexpression using the same pTRIPZ construct shown in (D). (G) EZH2 and DEK protein levels show a strong positive correlation in human primary invasive breast cancers. Pearson correlation = 0.50, P = 5.669 × 10 −6 . (H) EZH2 and DEK mRNA levels, detected by RNA-Seq, show a strong positive correlation in human primary breast cancers. Pearson correlation = 0.55, P = 2.21 × 10 −89 . Estrogen receptor (ER) negative samples, as determined by immunohistochemistry, are shown as orange circles whereas ER positive samples are in green. Blue and gray dots are samples with indeterminate or no data, respectively. Data for (G, H) are from the TCGA Firehose Legacy dataset for breast invasive carcinoma, accessed using www.cbioportal.org , and graphed using a log scale. Trend lines are shown in red. (I) EZH2 inhibition with GSK-126 limits hyperplasia in Dek-OE 3D organoids with no significant impact on control organoids. All organoids were imaged with a 20x objective lens and cropped to the same size. Organoid volume is graphed on the right and presented as mean ± SEM and statistical significance determined using a two-way ANOVA (genotype x treatment) for each day. There were no significant differences between any samples on day 3. Statistically significant differences were detected between groups on day 7. N = 3 mice (control) and N = 5 mice (Dek-OE) and primary tissue was collected from virgin adult females 8–12 wk old. Multiple organoids were quantified per donor. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. Source data are available for this figure.

Article Snippet: DEK (#610948, 1:1,000; BD Biosciences), SUZ12 (#3737, 1:1,000; Cell Signaling Technology), and EZH2 (#21800-1-AP, 1:2,000; Proteintech) primary antibodies were used to probe for co-immunoprecipitation.

Techniques: Gene Expression, RNA Sequencing, Control, Expressing, Residue, Western Blot, Immunohistochemistry, Staining, Knockdown, shRNA, Over Expression, Plasmid Preparation, Positive Control, Incubation, Immunoprecipitation, Construct, Inhibition

Journal: Life Science Alliance

Article Title: DEK promotes mammary hyperplasia and is associated with H3K27me3 epigenetic modifications

doi: 10.26508/lsa.202503230

Figure Lengend Snippet: (A) Graphical representation of the floxed Dek allele in a novel conditional Dek knockout mouse model. LoxP sites flank exons 3 and 4 which, when removed by Cre recombinase, create a premature stop codon in exon 5. Exon 1 (pink) of Dek is non-coding. (B) Dek flox mice were bred to CMV-Cre mice to create a whole-body knockout. Whole cell lysates from mammary glands collected from 5-wk-old virgin female DEK knockout mice show loss of Dek expression and lower levels of luminal marker cytokeratin 7 (CK7). Vinculin is used as a loading control. (C) Dek knockout females have smaller litter sizes than Dek WT and CMV-Cre − /Dek fl/fl controls. N values represent the number of separate dams in each Dam genotype group. N = 7 for Dek fl/fl and Dek +/+ controls, N = 12 for Dek +/Δ heterozygous dams, and N = 8 for Dek Δ/Δ knockout (Dek cKO) dams. Significance calculated with one-way ANOVA with dam genotype as the variable. (D) Kaplan-Meier survival curves reveal that half of pups born to Dek Δ/Δ knockout dams (N = 17 pups) die within 24 h with a few more not surviving until weaning at 28 d. Minimal loss of pups is observed in litters born to Dek fl/fl (N = 20 pups). Data were collected from litters born to at least three separate dams per dam genotype. Statistical significance was determined by log-rank test. *** P < 0.001. (E) Photographs of pups born to Dek WT (left) and KO (right) dams. Pups born to Dek WT dams had an observable milk spot within 24 h. Deceased pups from Dek KO dams were dehydrated and did not have a milk spot. (F) Whole cell lysates from virgin adult Dek Δ/Δ mammary glands had significantly less H3K27me3 levels than mammary glands from adult Dek fl/fl mice. (Bottom) Densitometry quantification of western blot data from (F) as determined by Image J. n = 4 per genotype. (G) DEK cKO mammary glands are smaller and have decreased branching density compared with WT mammary glands, as depicted by whole mount preparations (top). (Bottom) Branching density of Dek WT versus cKO mammary glands, as determined by Sholl analysis, is graphed. N = 3/genotype. All mice were virgin adult females. (H) p21 expression is increased, whereas Ezh2 expression, and H3K27me3 levels are lower, in the mammary epithelium of adult virgin Dek Δ/Δ mice compared with Dek proficient controls as determined by immunohistochemistry. n = 4–6/group. Quantification of staining intensity are graphed on the right and presented as mean ± SEM. For all studies, statistical significance is determined by an unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. Source data are available for this figure.

Article Snippet: DEK (#610948, 1:1,000; BD Biosciences), SUZ12 (#3737, 1:1,000; Cell Signaling Technology), and EZH2 (#21800-1-AP, 1:2,000; Proteintech) primary antibodies were used to probe for co-immunoprecipitation.

Techniques: Knock-Out, Expressing, Marker, Control, Western Blot, Immunohistochemistry, Staining

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: CRISPR-Cas9 screenings identified synthetic lethality co-mutations in ARID1A and PRC2 components (A) GeCKO lentivirus-library screening identified functional loss of genes that promote alcohol-mediated HCC development. (Middle) Lentiviral vector diagram for Cas9 and sgRNA for genome-scale knockout of coding sequences in human cells. (B) Identification of candidate genes needed for metastases. (C) Mutational frequencies of chromatin remodeling genes in alcohol- or/and HCV-associated HCCs. (D) GSEA identified PRC2 components (EZH2, EED, and SUZ12) were coenriched on NANOG-bound gene regions. (Bottom) Venn diagram of GEO datasets of HepG2 cells with wild-type ChIP-Seq data from mice liver TICs showed OXPHOS genes were coenriched with ARID1A and Nanog in ARID1A and SNF overexpressing datasets examining NANOG-bound regions. Data comparison from integrated analyses of ARID1A and SNF ChIP-Seq from HepG2 and NANOG ChIP-Seq data of TICs isolated from mice with alcohol-associated HCCs with ARID1A mutations. (E) Overlap of RNA profiling and Nanog ChIP-Seq data for NS5A transgenic and chronic alcohol-treated mouse liver TICs with ARID1A mutation showed PRC2 components (EZH2, EED, and SUZ12) were coenriched in NANOG-bound regions. Gene Set Enrichment Analyses (GSEA) identified PRC2 components EED, EZH2, and SUZ12 were co-enriched in NANOG-bound regions. (F) Genome-scale knockout, negative selection screening (GeCKO) in ARID1A-mutant HCCs that are resistant to sorafenib treatment. (Right) GeCKO screening for metastasis with single-guide RNAs (sgRNAs). (G) Synthetic lethality candidate genes (PRC2 genes) are listed. (H) Silencing ARID1A increased NANOG mRNA levels in TICs, but not in primary hepatocytes. Lipopolysaccharide (LPS) stimulation induces NANOG in TICs and sh-ARID1A-transduced primary hepatocytes, but not in primary hepatocytes. One of the major HCC risk factors is alcoholism that allows endotoxin leakage from intestinal tracts, leading to high endotoxin levels, stimulating NANOG transactivation. As LPS tolerance is evaded in TICs (not in primary hepatocytes), LPS stimulation induces NANOG. Star denotes statistical significance (p < 0.05, by Student T-test). (I) Hypothetical model of generation of TICs. Cancer-promoting mutations (ARID1A) increase PRC2 complex activity (including EZH2 component) and induce HCV/alcohol-mediated stem cell program (slow growing cells), leading to TIC-initiated HCC development. (J) Expression of HNF4A mRNA is correlated with ARID1A mRNA, but inversely correlated with PRC2 components (EZH2, EED, and AEBP2) in patients with HCC. RNA-seq from TCGA HCC patients were analyzed for the correlation of the expression between of HNF4A and ARID1A and that between HNF4A and PRC2 components EZH2, EED, and AEBP2. (K) Hypothetical model for interactions between ARID1A and PRC2 regulating TIC development. Alcohol intake changes gut microbiota to promote Gram(−) bacteria overgrowth in intestinal tracts and leaky gut, leading to endotoxin leakage from intestinal tracts. Endotoxin (LPS) in blood stream stimulates TLR4-CD14 complexes to transactivate NANOG to promote stem cell program. PRC2 complexes activate stemness gene and suppress anti-stemness genes (OXPHOS genes).

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: CRISPR, Library Screening, Functional Assay, Plasmid Preparation, Knock-Out, ChIP-sequencing, Comparison, Isolation, Transgenic Assay, Mutagenesis, Selection, Activity Assay, Expressing, RNA Sequencing, Bacteria

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: Nanog interacts with PRC2 components EZH2 and SUZ12 through carboxyl terminal domains (C1-W domains) (A) Schematic illustration of NANOG deletion constructs containing an N-terminal Flag tag. (B) Schematic illustration of EZH2 deletion constructs containing an N-terminal Myc-tag. Upper panels: Immunoblots are shown following immunoprecipitation with anti-Myc or anti-Flag and immunoblotted with anti-Flag or anti-Myc, respectively. Lower panels: Immunoblots of whole cell lysates with anti-Flag or anti-Myc. Lane numbers correspond to constructs shown in (A). (C) HEK 293T cells were cotransfected with the indicated Myc-tagged EZH2 deletion constructs and Flag-tagged NANOG full-length constructs. Upper and lower panel schema are the same as in (B). EZH2 and SUZ12 bind W-domains of NANOG. (D) HEK 293T cells were cotransfected with Flag-tagged hNANOG full-length expression plasmids and Myc-tagged hSUZ12 expression constructs as indicated. Half of cell lysates were further treated with DNaseI and sonication, then both portions were subjected to coimmunoprecipitation by using either anti-Flag antibody (IP) or an isotype matched IgG negative control (IgG). The immunoprecipitates or input cell lysates as a control (WCL) were analyzed by SDS-PAGE and immunoblotted (IB) with antibodies against Myc or Flag epitope tag. (E) Schematic representation of EZH2 mutant expression vectors that were used for IP-western blot analyses to examine the minimal domains for interaction between NANOG and EZH2. (F) IP-western blots showed that NANOG and EZH2 interact through H2-CYS domains of EZH2. Upper: Cell lysates were immunoprecipitated with anti-Flag or anti-Myc, as indicated then immunoblotted with anti-Myc or anti-Flag, respectively. Lane numbers correspond to gene constructs shown in (E). Lower: Whole cell lysates were analyzed after transfection and immunoblotted with anti-Myc or anti-Flag as indicated. (G) Upper: Gene constructs for SUZ12. Lower: Immuoprecipitates of cell lysates as indicated with anti-Myc or anti-Flag followed by immunoblotting with anti-Myc or anti-Flag. IP-western blots showed that NANOG and SUZ12 interacted through H2-CYS domains of EZH2.

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Construct, FLAG-tag, Western Blot, Immunoprecipitation, Expressing, Sonication, Negative Control, Control, SDS Page, Mutagenesis, Transfection

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: PRC2 component proteins (EED, SUZ12, and EZH2) interact with NANOG to stabilize NANOG (A) Schematic of NANOG deletion constructs containing an N-terminal Flag tag. These deletion mutants of NANOG were tested for EED binding. (B) HEK 293T cells were cotransfected with the indicated Myc-tagged EED full-length constructs and various Flag-tagged NANOG deletion constructs as listed in (A) and as blot lane numbers. Cell lysates were immunoprecipitated with anti-Myc or anti-Flag followed by immunoblotting with anti-Flag, as indicated. Nanog interacts with PRC2 components EED through carboxyl terminal domains (C1-W domains). Note NANOG with C1-W domain loses this interaction. (C) Reciprocal IP-western blot analyses confirmed interactions between NANOG and EED. HEK 293T cells were cotransfected with the indicated Myc-tagged NANOG deletion constructs and Flag-tagged EED full-length constructs. Immunoprecipitation and immunoblotting were conducted with antibodies as indicated in the immunoblots. Different deletion mutants of NANOG were tested for EED binding. Note that NANOG N-terminus and C1-W domain is needed for interaction with EED. (D) The EED silencing reduced NANOG protein levels in TICs. Student’s T-test was used for statistical analyses. Star marks denote significantly different (p < 0.05). (E) RT-qPCR analyses of TICs with EED-knockdown. Silencing of EED did not alter NANOG mRNA levels (Left) while silencing of EED induced COX6A2 expression (Left), but reduced OCT4 and SOX2 mRNAs. (F) Spheroid colony formation assay. Upper-EED expression transformed p53-deficient hepatoblasts and was potentiated by NANOG. Lower-HNF4A transcription was unaffected by NANOG; however, EED repressed HNF4A transcription. Stars denote statistical significance (p < 0.05). (G) EED silencing reduced protein stability of NANOG. Huh7 cells were transduced with scrambled shRNA (sh-scr) or shRNA targeting EED. Seventy-two hours after transduction, cycloheximide (CHX) was added and cells were harvested at the indicated times. NANOG protein level was detected by immunoblotting (Top panel). Results of a representative experiment (n = 3) are plotted as percentage of starting NANOG protein level for half-life determination (Bottom panel). (H) IP-western blots of EED binding to NANOG mutants. CD133(−) non-TICs and TICs were lysed and immunoprecipitated with anti-NANOG antibody and examined for immunoblot analyses. Endogenous EED interacts with NANOG. Huh7 cells were transfected with shRNA for EED followed by immunoblotting with anti-NANOG or anti-EED as indicated. Cells silenced for EED did not co-precipitate NANOG as was observed for control sh-scrambled treated cells. As shown, TICs associated with the three subunits of PRC2 (EED, EZH2, and SUZ12), RbAp46/48, JARID2, and AEBP2. (I) Hypothetical mechanisms of EED-NANOG interaction promoting stabilization and activation of NANOG signaling in TICs. EED may stabilize NANOG through its dependence on PEST domain phosphorylation allowing homodimerization. Different deletion mutants of NANOG were tested for EED binding. Note that NANOG N-terminus and C1-W domain is needed for interaction with EED. Created with biorender.com (Agreement Number: CN259LCOTO). (J) (Left) sh-EED and sh-Scrambled Huh7 cells were transfected with flag-tagged NANOG vector for 48 h. At 24 h post-transfection, some cells were treated with 10 μM MG132. Whole cell lysates were used in western blot to quantify Flag-tagged NANOG. EED silencing reduced NANOG protein levels. Proteasome inhibitor (MG132) treatment enhanced NANOG protein levels that were accentuated by EED silencing. (Right) Immunoprecipitation-western blot analysis of NANOG in EED knockdown Huh7 cells. The sh-EED and sh-scrambled-transduced Huh7 cells were lysed with RIPA buffer. The cell lysates were pre-cleared with magnetic Protein A beads then incubated with anti-Nanog antibody overnight. The immunocomplex was pulled down with Protein A beads followed by western blot analysis of NANOG. Blot was prepared and analyzed by ImageJ. (K, Top) The immunocomplex was pulled down with Protein A beads followed by western blot analysis of FBXW8. Immunoprecipitation-western blot analysis of FBXW8 in EED knockdown Huh7 cells. Co-IP western blot analysis of NANOG and its PEST domain-associated proteins. Sh-EED and sh-scrambled Huh7 cells transfected with Flag-NANOG vector were treated or untreated with 10 μM MG132. The cell lysates were pre-cleared with magnetic Protein A beads then incubated with anti-Flag antibody overnight. The immunocomplex was pulled down with Protein G beads followed by western blot analysis of FBXW8 and PIN1 proteins. Western blot analysis of FBXW8, ubiquitin, and PIN1 was done to determine their association with NANOG under these conditions. Sh-EED and sh-scrambled Huh7 cells co-transfected with Flag-NANOG and MYC-FBXW8 vector were treated or untreated with 10 μM MG132 (proteasome inhibitor). Following the same immunoprecipitation procedure, the lysate was immunoblotted with anti-Myc antibody. IP Flag-NANOG/WB FBXW8 is for detection of FBXW8. The next line is for WB PIN1. (K, Bottom) The same experimental procedure as described above. EZH2, EED, and β-Actin proteins were detected by western blot analysis. (L) EED knockdown and sh-scrambled Huh7 cells were cultured, and some were treated with 10 μM MG132 for 24 h. Cells were lysed and total RNA collected. The RNA was reverse transcribed to cDNA and subsequently used in quantitative-PCR with SYBR green. Oligonucleotides for NANOG and COX6A2 were used to amplify the respective gene. (M) EED-NANOG cooperation in tumor development. Tumor growth after TIC subcutaneous transplantation in NSG mice was markedly suppressed by EED KD but partially rescued by concomitant expression of NANOG. NANOG alone enhanced tumor growth which was attenuated by EED KD below the growth observed in control TICs. (N) EED KD reduced NICD-induced tumor growth by p53−/− hepatoblasts orthotopically transplanted into the livers of NSG mice. (O) Screening for selective inhibitors for SUZ12-NANOG interaction. Diagram of drug screening by fluorescence polarization assays. (P) XTT cell viability assays in CD133(+) and CD133 (−) Huh7 cells. Note NSC8090, NSC14540, and NSC123127 (Doxorubicin) are selectively cytotoxic to CD133+ cells. Small molecule NSC8090 selectively kills CD133+ Huh7 cells. “: p < 0.05. (Q) NSC8090 blocks EED-NANOG interactions in CD133+ Huh7 cells. (R) IC 50 values of combination treatments of a PRC2-NANOG inhibitor and/or sorafenib were examined in different HCC cell lines in the presence or absence of ARID1A mutations. Stars denote statistical significance.

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Construct, FLAG-tag, Binding Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Colony Assay, Transformation Assay, Transduction, shRNA, Transfection, Control, Activation Assay, Phospho-proteomics, Plasmid Preparation, Incubation, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transplantation Assay, Drug discovery, Fluorescence

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: Silencing EZH2 reduced self-renewal ability and tumorigenesis, especially in ARID1A-silenced cells (A) Silencing EZH2 reduced spheroid formation. (B) Silencing EZH2 increased cell sizes and induced morphological changes. (C) Silencing EZH2 induced epithelial phenotypes. Stars denote statistical significance. (D) Ezh2 inactivation reduced tumor growth in NSG mice. sh- Ezh2 -knockdown reduced tumor sizes engrafted in NSG mice. (E) Tumor weight reduction occured after sh- Ezh2 -knockdown. (F) NANOG transformed LPCs with Ctnnb1 activation or TP53/Arid1a inactivation in orthotopic injection mouse models. ∗: p < 0.05, Student T-test. (F, G) LPCs were tested in the presence or absence of NANOG transduction in sh- ARID1A , p53 R270H or constitutively active β-Catenin-expressing cells. Representative tumors are shown for indicated gene knockdowns or overexpression. (G) Red fluorescence (dsRed)+ tumors in liver and lung. Arid1a knockdown plus NANOG expression promoted dsRed+ lung metastasis. (H) Tumor progression and poor survival rate in patients with HCC with high expression of NANOG and EZH2. The Kaplan-Meier plot was generated from TCGA liver cancer (LIHC) gene expression profiles and corresponding survival data in a total of 370 patients by evaluation for top and bottom 20% expression levels of NANOG and EZH2. (I) Liver tumor compared to surrounding normal tissue from patients with HCC; stained for both NANOG and EZH2. (I, Bottom) NANOG and EZH2 of the PRC2 are expressed in human HCC, especially in metastatic HCC.

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Knockdown, Transformation Assay, Activation Assay, Injection, Transduction, Expressing, Over Expression, Fluorescence, Generated, Gene Expression, Staining

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: PRC2 complexes and PAPRδ competes the W-rich domains of NANOG (A) (Upper Left) Schematic of PPARδ truncated expression constructs with c-Myc epitope tag. NANOG binding activity is indicated. (Lower Left) Hypothetical model of FAO inhibitor-mediated apoptosis in TICs. NANOG promotes FAO in HCC cells. (Upper Right) IP-western blot analyses between NANOG and PPARδ truncation mutants. The N-terminus of NANOG (aa1-72) binds PPARδ. (Lower Right) Confirmation of PPARδ truncation mutant expression. Lane numbers correspond to constructs shown in schematic. Numbers on the top of immunoblots denote which PPARδ mutant was combined with full-length NANOG (FL). (B) Left-diagram of Flag-NANOG constructs used for testing PPARδ binding activity. Right- Co-IP combinations with NANOG mutants and Flag-PPARδ mutants; lane numbers correspond to latter mutants. IP-western blots showed that NANOG and PPARδ interacted through W domains of NANOG. Numbers on the top of immunoblots denote which NANOG mutant was combined with full-length PPARδ (FL). (C) Hypothetical mechanisms of EED-NANOG interaction promoting stabilization and transcriptional suppression by NANOG-PRC2 interactions in TICs. EED may stabilize NANOG through its dependence on PEST domain phosphorylation. PKCε-mediated phosphorylation of T200 of NANOG promotes homodimerization. Transcriptional suppressor PRC2 complexes (EZH2-SUZ12) and transcriptional activator PPARδ-NANOG share binding-sites and possibly compete with W domain of NANOG. (D) Hypothetical model of FAO inhibitor-mediated apoptosis in TICs. NANOG suppresses mitochondrial respiration by suppressing OXPHOS genes, but transactivates fatty acid oxidation genes (FAO) by transcriptional activation through competing out PRC2 complex binding in tryptophan (W)-rich domain that is used for NANOG dimerization and activation as well. The TCA cycle generates NADH. The transfer of reducing equivalents from NADH to NADP+ via nicotinamide nucleoside transhydrogenase (NNT) provides the reducing potential energy (NADPH). NADPH reduces glutathione and controls mitochondrial ROS. (Right) Inhibition of FAO inhibits energy production in TCA cycle and OXPHOS and promotes ROS production, leading to cytochrome c release and apoptosis. Loss of mitochondrial membrane potential Δψ promotes apoptosis of TICs via BAX/BAK oligomerization. (E) Changes in HNF4A and c-Myc mRNAs in response to ARID1A-1 or ARID1A-2 silencing. RT-qPCR analysis for ARID1A knockdown in Huh7 cells decreased ARID1A and HNF4A but increased Myc mRNA level. Stars denote statistical significance (p < 0.05, Student T-test). (F) Cytosolic and mitochondrial marker expression. Left-Huh7 cells treated with shRNA against scrambled or ARID1A were treated with FAOi and EZH2i for 1 h, then cell lysates were harvested at time points as indicated. Cells were fractionated into cytoplasmic (left panel) and mitochondrial fractions (right panel), followed by SDS-PAGE and immunoblotting with antibodies against cytC, Bak, Bax, Cu/ZnSOD, or VDAC, as indicated. (G) Knockdown of ARID1A alleviated apoptotic activity of FAOi and EZH2i. Protein expression levels for cytochrome c and BAX were measured. The densitogram of immunoblots shown in panel E (left) was quantified by ImageJ.

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Expressing, Construct, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay, Phospho-proteomics, Activation Assay, Inhibition, Membrane, Quantitative RT-PCR, Knockdown, Marker, shRNA, SDS Page

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet: EZH2 inhibition derepresses NANOG target gene through downregulation of repressive histone marks (H3K27me) and increases activation histone marker (H3K27ac) (A) FACS measurement of ROS production in Huh7 cells. Both FAOi and EZH2i promoted mitochondrial ROS levels and were compared for single, or combination FAOi and EZH2i treatments. (B) Loss of ARID1A enriched label retaining (slow-cycling) cell subpopulation and reduced mitochondrial ROS production. Huh7 cell stably expressing control shRNA or shRNA against ARID1A were treated with vehicle, Etomoxir, GSK126, or combination of Etomoxir and GSK126. Cells were stained with CellTrace Violet (label retaining population for which intensity is reduced at every cell division) and DiOC 6 (mitochondria membrane potential) and subjected to flow cytometry analysis. (Right) Quantification of percentage of Dioc 6 positive population (Top panel) and quantification of percentage of CellTrace Violet positive but Dioc 6 negative population (Bottom panel) in cells treated with sh-scrambled versus ARID1A-scilenced Huh7 cells. Purple-colored (Top) or Red-colored quadrants (Bottom) were compared in FACS graphs. (C) EZH2 inhibition derepresses NANOG target through downregulation of repressive histone marks (H3K27me) and increases activation histone marker (H3K27ac). (D) COX6A2 gene was upregulated upon FAOi and EZH2i treatment. Huh7 cells were treated singly or in combination with FAOi and EZH2i. RT-qPCR was performed to detect mRNA level of COX6A2 after FAOi and EZH2i treatments. ∗: Statistical significance (p < 0.05 by Student’s T-test). (E) EZH2 inhibition derepress OXPHOS NANOG target and increases activation histone marker (H3K27ac). ChIP-qPCR was performed to show the fold enrichment of anti-H3K27Me3 (Left) or anti-H3K27Ac binding (Right) on COX6A2 promoter region. The site examined corresponds to the promoter proximal NANOG binding site of the COX6A2 gene. ∗: Statistical significance (p < 0.05). (F) ChIP-qPCR was performed to show the fold enrichment of anti-NANOG, anti-EZH2, or anti-EED binding on COX6A2 promoter region. Stars denote statistical significance (p < 0.05, Student T-test). (G) Hypothetical model of NANOG-mediated gene suppression through NANOG-PRC2 interactions. PRC2 complexes and PPARδ activators compete NANOG tryptophan (W)-rich domain of NANOG for global transcriptional suppression or activation. PRC2 component EED and ubiquitin E3 ligase FBXW8 compete NANOG phosphodegron sequence PEST domain (P, E, S, and T-rich phosphodegron sequence) for stabilization or E3 ligase FBXW8-dependent degradation. Inhibitors of NANOG-PRC2 interactions with compete NANOG phosphodegron domain (PEST domain) to destabilize NANOG protein to inhibit self-renewal abilities of TICs and reduce tumor growth in humanized mice. Therefore, PRC2 components stabilized and differentially regulated NANOG target genes which depend on the NANOG W domain-binding partners (PPARδ or EZH2/SUZ12). These mechanisms would be conserved in many different TICs or embryonic stem cells.

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Inhibition, Activation Assay, Marker, Stable Transfection, Expressing, Control, shRNA, Staining, Membrane, Flow Cytometry, Quantitative RT-PCR, ChIP-qPCR, Binding Assay, Ubiquitin Proteomics, Sequencing

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet:

Article Snippet: Human Recombinant EZH2, GST tagged, full length , Creative BioMart , EZH2-285H.

Techniques: Control, Virus, Recombinant, Modification, Reverse Transcription, SYBR Green Assay, Cell Culture, Gel Extraction, Plasmid Preparation, Software

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: The association of EZH2 and JMJD3 expressions with HSC activation and liver fibrosis. ( A ) The expressions of EZH2, JMJD3, α-SMA, H3K27me2/3 and β-Actin as inner control during culture-induced activation of rat primary HSCs (0, 1, 3, 5, 7 days) was analyzed by Western blot. ( B ) In the gene arrays dataset for liver biopsy samples from 124 liver fibrosis patients (GSE84044, ref 29), linear regression was used to analyze the association of hepatic expression of PRC2 subunit genes ( EZH2 , SUZ12 , EED and RBBP4 ) with sequential histological staging of fibrosis, Scheuer score, S , which is divided as S0 (stage 0, n = 43), S1 ( n = 20), S2 ( n = 33), S3 ( n = 18) and S4 ( n = 10). The box plot and fitting curve showed the association trend. The significance of correlation was expressed as r -squared values of regression and P < 0.01 was considered as statistical significance.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Activation Assay, Western Blot, Expressing

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: EZH2 and JMJD3 regulate HSC activation through modulating Tgfβ1 signaling pathway. Rat primary HSCs were cultured and treated with inhibitors of EZH2 (DZNep) or JMJD3 (GSK-J4), or transfected with siRNAs of Ezh2 , or infected with adenovirus recombined with Jmjd3 . RNA-seq was conducted for HSCs treated with DZNep or DMSO as control. ( A ) HSCs growth and morphology were assessed in fifth day after administration, the magnification is 200×. ( B-D ) EZH2, JMJD3, α-SMA and COL1A expressions and H3K27 methylation level were determined by Western blot with β-ACTIN and Histone 3 as inner controls respectively. * P < 0.05. ( E ) Basing on the DEGs associated with DZNep treatment in HSCs, the upstream regulator analysis of IPA identified Tgfβ1 among the most significantly inhibited growth factors or cytokines. The top candidates are shown with overlap P value < 0.01 and | activation Z -score | >2. (F) The heatmap shows transcriptional value log 10 RPKM for genes encoding effectors, activators and inhibitors of TGFβ1 signaling pathway. Bambi in the inhibitor panel was highlighted in red.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Activation Assay, Cell Culture, Transfection, Infection, RNA Sequencing Assay, Methylation, Western Blot

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: Cellular phenotypic effects of EZH2 inhibition with DZNep or GSK126 on HSCs cell cycling, proliferation, senescence and apoptosis. JS1 cells were administrated with DZNep, GSK126 or DMSO and subjected to analyses of cell cycle ( A ), cell growth ( B-C ), senescence ( D ) and apoptosis ( E-F ) with staining and flow cytometry. The transcriptional expression of cell proliferation marker MKI67 gene in rat primary HSC, human LX-2 and mouse JS cells were measured by RT-qPCR (C). Cell senescence was determined by measuring SA-β-Gal activity (D). The apoptotic cells were determined by Annexin-V-PI double staining (E) and FITC-dUTP staining by TUNEL apoptosis assay (F).The yellow frames in panel E present early apoptotic cells. Data were measured from triple independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Inhibition, Staining, Flow Cytometry, Expressing, Marker, Quantitative RT-PCR, Activity Assay, Double Staining, TUNEL Assay, Apoptosis Assay

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: DZNep treatment results in reduced hepatic expressions of fibrotic marker genes in mice with CCl 4 -induced liver fibrosis and prevalent downregulation of ECM component genes in HSCs. Hepatic expressions of H3K27me3, Histone3, EZH2 and β-Actin in CCl 4 mice with DZNep or DMSO treatment were determined by Western blot ( A-B ). Hepatic transcriptions of Ezh2 , α-Sma , Col1a , Mmp2 and β-Actin were determined by RT-qPCR. The pre-ranked gene list for transcriptomic profiling of rat primary HSCs treated with DZNep or DMSO was subjected to GSEA, ECM genes were significantly enriched ( C ). The heatmap shows transcriptional changes of various ECM component genes ( D ). Statistical significance was determined with Student's t -test in independent samples.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Marker, Western Blot, Quantitative RT-PCR

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: HSC-specific Ezh2 silencing in vivo ameliorated liver fibrosis. ( A ) Construction of amiRNA vector for Ezh2 silencing. The amiRNA consisted of miR-30a backbone as structural scaffold and two concatenated pre-miR30-shRNA cassettes embedding valid interfering shRNA (amiR- Ezh2 ) or negative control shRNA (amiR-NC) as cargos. Based on lentiviral microRNA expressional vector, amiRNA was inserted into the cloning site located in EGFP intron, the original CMV promoter was substituted with human GFAP promoter. ( B ) Ezh2 silencing in JS1 cells was achieved by infection of lentiviral-amiR- Ezh2 , expressions of EZH2, COL1A, α-SMA and β-ACTIN were determined by Western blot. ( C ) In CCl 4 mice, lentivirally expressed amiRNA was transduced into the liver through tail-vein injection, therapeutic effects of amiRNA-based HSC-specific Ezh2 silencing was analyzed by measuring collagens deposition with Masson's trichrome staining and α-SMA expression with immunohistochemistry.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: In Vivo, Plasmid Preparation, shRNA, Negative Control, Clone Assay, Infection, Western Blot, Injection, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis

doi: 10.7150/thno.46360

Figure Lengend Snippet: EZH2 and JMJD3 regulate Bambi , Cdkn1a , Gadd45a and Gadd45b. Primary rat HSCs were treated with DZNep or infected with adenovirus- Jmjd 3 or their controls respectively. The effects of DZNep on H3K27me3 ( A ) or Jmjd3 overexpression on H3K27me2/me3 ( B ) enrichments at target genes were determined by ChIP-qPCR. The 5′ endpoints of PCR products are positioned as upstream (-) or downstream (+) from transcription start sites. The effect of DZNep treatment or Jmjd3 overexpression on transcript and protein products of target genes were determined by RT-qPCR ( C-D ) and Western blot ( E-F ) respectively. β-Actin was used as internal control. Hepatic transcriptions of Bambi and Cdkn1a in CCl 4 ( G ) and BDL ( H ) mice were determined by RT-qPCR. * P < 0.05, ** P < 0.01.

Article Snippet: We obtained from Addgene the mouse Ezh2 retroviral vector, MSCVhygro-F- Ezh2 (cat. no. 24926) and the enzymatically inactive mutation vector, MSCVhygro-F- Ezh2 -F667I (cat. no. 24927) , the plasmid pCS2- Jmjd3 -F (cat. no. 17440) containing full-length mouse Jmjd3 cDNA, and hereby generated a demethylase activity defective Jmjd3 mutant mutating histidine 1388 to alanine (H1388A) with point mutation method .

Techniques: Infection, Over Expression, Quantitative RT-PCR, Western Blot

Journal: Nature Communications

Article Title: The non-coding RNA landscape of human hematopoiesis and leukemia

doi: 10.1038/s41467-017-00212-4

Figure Lengend Snippet: LINC00173 is a novel regulator of granulocytic development. a – d RNAi (shRNA)-mediated knockdown of LINC00173 in CD34 + HSPCs in vitro. a Granulocytic in vitro differentiation (day 14). Upper panel: May-Grünwald Giemsa ( MGG ) staining; scale bars 20 µm. Middle panel: neutrophil peroxidase ( POX ) staining; scale bar 20 µm. Lower panel: flow cytometric analysis of CD66b and CD13 surface marker expression. The bar graphs (right) show the mean ± s.d. of three independent experiments. b Percentage of bead-positive cells in a phagocytosis assay. The histogram depicts the fluorescence intensity. c Number of BFU-E and CFU-G/M (CFU-my) in methylcellulose-based CFU-assays normalized to the non-silencing shRNA control ( ctrl ). d Number of shRNA-transduced cells during granulocytic in vitro differentiation normalized to day 0. e Ratio of RFP657 + sgRNA-transduced vs. untransduced cells relative to the non-targeting control (sgRNAs against luciferase), using monoclonal NB4 cell lines stably expressing dCas-KRAB ( n = 6). f Cytoplasmic to nuclear ratio of LINC00173 determined by qRT-PCR on fractionated RNA from THP-1 cells. g RNA FISH with tiled biotinylated probes in THP-1 cells; scale bars 10 µm. h GSEA results for 52 hematopoiesis-associated gene sets upon LINC00173 knockdown in CD34 + HSPCs. The plot shows normalized enrichment scores ( NES ) against nominal P -values of the normalized enrichment score , dotted line: P = 0.05. i Heatmap showing expression of leading-edge genes from the “Fischer_DOWN IN SEVERE APLASTIC ANEMIA” gene set across the human DMAP data set . j RIP in NB4 cells using two different antibodies, followed by qRT-PCR to detect binding of EZH2 to LINC00173 . Data are presented as percent of input in comparison to B2M . k ChIP-seq density heatmaps for H3K27me3 in promoter regions of leading-edge genes from the indicated gene sets upon LINC00173 knockdown. shRNA-transduced CD34 + HSPCs (sh-L173 and sh-CTRL) are compared to untransduced and uncultured CD34 + HSPCs. Clusters of promoters with differential H3K27me3 marks are highlighted. Representative stem cell-specific genes are shown (right). Data are presented as mean ± s.d. a – f , j , or s.e.m. c , e . * P < 0.05; ** P < 0.01; ns not significant; P -values were calculated using one-way ANOVA with Dunnett’s post hoc test

Article Snippet: Dynabeads protein G (Invitrogen) were then washed twice using a Dynal magnet (Invitrogen) with 0.5 ml citrate-phosphate buffer (pH 5), and resuspended in citrate-phosphate buffer with the following antibodies: Anti-EZH2 polyclonal rabbit antibody (07-689; Millipore), Anti-Ezh2 (D2C9) XP rabbit antibody (Cell Signaling), and rabbit (DA1E) mAb IgG XP Isotype Control #3900 (Cell Signaling).

Techniques: shRNA, Knockdown, In Vitro, Staining, Marker, Expressing, Phagocytosis Assay, Fluorescence, Control, Luciferase, Stable Transfection, Quantitative RT-PCR, Binding Assay, Comparison, ChIP-sequencing